www.elsevier.com/locate/gene

Gene 315 (2003) 103–112

Interaction of proteins with promoter elements of the human U2 snRNA

genes in vivo

Diana C. Boyd, Ana Pombo1, Shona Murphy*

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

Received 17 March 2003; received in revised form 9 May 2003; accepted 22 May 2003

Received by D. Higgs

Abstract

The multicopy human U2 small nuclear (sn)RNA genes are transcribed by RNA polymerase (pol) II and contain two major promoter

elements upstream of the transcription start site: an essential proximal sequence element (PSE) at around �55 and a distal sequence element

(DSE) at around �220. We have carried out an in vivo footprinting analysis on these genes, and the results suggest that most, if not all, of the

U2 gene promoters are bound by factors in interphase. Both the DSE and the PSE are protected from digestion, and the pattern of methylation

protection over the DSE is virtually identical to that obtained in vitro using nuclear extract. Our results also indicate that the DNA between

the PSE and the transcription start site is distorted and that proteins interact with the promoter between �20 and �33. Mutation of this

sequence affects both the accuracy of initiation and polymerase specificity, underlining the importance of this region in U2 gene expression.

We have also analysed the pattern of protection over the DSE and PSE of the U2 genes in mitotic cells. The degree of protection over all

promoter elements is drastically reduced, suggesting that loss of DNA binding factors from the promoter plays a role in the shutdown of U2

gene transcription in mitosis.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Mitosis; Nucleosome; Oct-1; Pol II; PTF; Sp1

1. Introduction

The human small nuclear (sn)RNA genes transcribed by

RNA polymerase II (pol II) (e.g., U1 and U2) contain one

essential promoter element, the proximal sequence element

(PSE), located around �55, and an enhancer-like distal

sequence element (DSE) around �220, which activates

transcription by about 100-fold. Promoters of snRNA

0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0378-1119(03)00717-0

Abbreviations: A, adenosine; bp, base pair(s); C, cytidine; DMS,

dimethyl sulphate; DNase, deoxyribonuclease; DSE, distal sequence

element; G, guanosine; kb, kilobase(s); min, minute(s); N, any nucleoside;

nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PCR, polymerase chain

reaction; pol, polymerase; PSE, proximal sequence element; sn, small

nuclear; T, thymidine.

* Corresponding author. Tel.: +44-1865-275616; fax: +44-1865-

275556.

E-mail address: shona.murphy@pathology.ox.ac.uk (S. Murphy).1 Present address: MRC-Clinical Sciences Centre, Imperial College

School of Medicine, Hammersmith Hospital Campus, Du Cane Road,

London W12 0NN, UK.

genes transcribed by pol III (e.g., U6 and 7SK) contain

the same elements in the promoter but have an additional

TATA sequence at �25 that plays a major role in poly-

merase specificity (reviewed by Hernandez, 2001).

Several factors required for transcription of pol II-

dependent snRNA genes have been identified. The DSE

often contains binding sites for the ubiquitously expressed

transcription factor Oct-1, which can activate transcription

of both pol II and pol III-transcribed snRNA genes in

vivo in addition to protein-coding genes (Murphy, 1997).

The DSE of the U2 genes also contains binding sites for

SP1 (Janson et al., 1987), which is another factor com-

monly required for transcription of protein-coding genes.

The PSE is recognised by a multisubunit snRNA gene-

specific transcription factor known as PTF, PBP or

SNAPC (reviewed by Hernandez, 2001). The POU DNA

binding domain of Oct-1 interacts directly with PTF to

potentiate its binding to the PSE (Mittal et al., 1996;

Murphy et al., 1992; Murphy, 1997), and a mutation that

abrogates this interaction reduces the ability of Oct-1 to

activate transcription in vivo (Murphy, 1997).

U25VPrimer 1 5V-TGGCTCGATACGAACAAGGAAGT-3V

(�334 to �312)

Primer 2 5V-CCCGGCTCCCCCAGGCAGA-3V(�297 to �279)

Primer 3 5V-AGGCAGAGGCGGCCCCGGGGGCGGA-3V(�285 to �261)

U2PSE

Primer 1 5V-GGAGAATAAGAAATCAGCCCGAG-3V(�151 to �129)

Primer 2 5V-CAGCCCGAGAGTGTAAGGGCGTC-3V(�137 to �115)

Primer 3 5V-GCCCGAGAGTGTAAGGGCGTCAATAGCGC-3V(�135 to �107)

U23VPrimer 1 5V-CACTTGATCTTAGCCAAAAGGCC-3V

(+33 to +11)

Primer 2 5V-GCCAAAAGGCCGAGAAGCGATG-3V(+21 to �1)

Primer 3 5V-GCCAAAAGGCCGAGAAGCGATGCGC-3V(+21 to �4)

Long linker 5V-GCGGTGACCCGGGAGATCTGAATTC-3V

D.C. Boyd et al. / Gene 315 (2003) 103–112104

In common with mRNA genes the pol II-dependent

snRNA genes require TBP, TFIIB, TFIIA, TFIIE and TFIIF

for transcription (reviewed by Hernandez, 2001) although

the complete composition of the preinitiation complex

remains to be determined. The TBP/TAFII complex TFIID

involved in transcription of mRNA-coding genes cannot

substitute for TBP in transcription of the human U1 gene in

vitro (Bernues et al., 1993) and the form of TBP used is

unclear. However, at least one TAFII is associated with U2

genes (Christova and Oelgeschlager, 2002). The recruitment

of TBP to these genes may be facilitated by interaction with

PTF (Henry et al., 1995; Sadowski et al., 1996; Yoon and

Roeder, 1996).

Although the promoter elements of several of the pol II-

transcribed snRNA genes have been analysed in some detail

by mutational analysis, it is not known how factors interact

with the promoters in living cells. We have therefore carried

out a comprehensive in vivo footprinting analysis using

dimethyl sulphate (DMS), deoxyribonuclease (DNase) I and

micrococcal nuclease to determine the architecture of the

promoters of the multicopy, tandemly repeated U2 genes in

vivo. In unsynchronised HeLa cells, both the PSE and the

DSE are strongly protected from methylation and digestion,

suggesting that most of the U2 gene promoters are occupied

by factors in interphase. The DNA between the DSE and

PSE is protected from digestion by micrococcal nuclease

and shows regular sites of hypersensitivity to DNase I,

suggestive of a nucleosome. Protection from micrococcal

nuclease digestion also extends across the PSE and down-

stream past the transcription start site. Furthermore, the

sequence between �33 and �20, located between the PSE

and the start site of transcription, is protected from methyl-

ation and DNAse I digestion, indicating that a factor(s)

interacts with this region of the promoter. Mutation of this

sequence, which is similar to the same region of the U1

promoter, affects initiation and polymerase specificity. In

addition, we show that binding of factors to the promoter is

severely reduced in mitosis, when transcription is generally

extinguished.

Short linker 5V-GAATTCAGATC-3VLinker primer 5V-GAATTCAGATCTCCCGGGTCACCGC-3V

2. Materials and methods

2.1. In vivo footprinting

For all footprinting studies, 1.5�10�6 cells were seeded

16 h before treatment. Treatment of cells by DMS and

nuclei by DNase I, DNA preparation and linker-mediated

polymerase chain reaction (PCR) were carried out exactly as

described by Boyd et al. (2000) using the primers described

below. For linker-mediated PCR of chromatin treated with

micrococcal nuclease, cells were treated with 100 U micro-

coccal nuclease (Boehringer Mannheim) for 5 min at room

temperature, in 200 Al physiological buffer containing 0.3%

NP40 (Boyd et al., 2000). The reaction was stopped with

500 Al lysis buffer [450 mM NaCl, 75 mM Tris–HCl pH 8,

37.5 mM EDTA, 0.3% SDS and 0.3 mg/ml proteinase K

(Boehringer Mannheim)] and DNA purified as described by

Boyd et al. (2000). To prepare control purified naked DNA,

50 Ag DNAwas treated with 1 U micrococcal nuclease for 5

min at room temperature in 1 ml final volume containing 25

mM Tris–HCl pH 7.5, 5 mM MgCl2, 50 mM NaCl and 2.5

mM CaCl2. The reaction was stopped with 50 Al 0.5 M

EDTA and 50 Al 5 M NaCl, before extracting twice with

phenol/chloroform and ethanol precipitating. Both in vivo

and control DNA samples were then phosphorylated with

2.5 U polynucleotide kinase (Boehringer Mannheim) in 1�commercial buffer containing 150 mM ATP before ligation

of linkers. DMS treatment of mitotic cells was carried out as

described by Boyd et al. (2000) after preparation by mitotic

shake off as described by Pombo et al. (1998). The

percentage of cells in mitosis was assessed by microscopic

examination both with and without DAPI staining and was

89% in the cells used for the experiment of Fig. 6.

Nested primers for linker-mediated PCR were as follows:

2.2. S1 nuclease and primer extension

For each 90-mm dish of HeLa cells, 5 Ag of each U2

construct was co-transfected with 500 ng of a plasmid

containing the pol III-transcribed Adenovirus VA1 gene

using Lipofectamine (Gibco) as recommended by the

manufacturers. a-Amanitin (Calbiochem) was added to the

cells to a final concentration of 5 Ag/ml 12 h before harvest-

ing, where indicated. After transfection, total RNA was

prepared as described by Medlin et al. (2003). S1 analysis

was carried out as described by Murphy (1997) using the

same DNA oligo for the U2 maxi clones, the oligos 5V-ATGATACCCTTGCGAATTTATCCACCAGACCACG-

GAAGAGTGCCCGCTTAC-3V for VAI and 5V-GCGAT-

D.C. Boyd et al. / Gene 315 (2003) 103–112 105

CAATGGGGTGACAGAACAAGCTTAGTGTCGCAGC-

CAGATCGCCCTCACATCCAGCGATGCGTCGCCTTC-

3Vfor U2/7SK RNAs. The products of S1 analysis are 40 and

63 nt for the VAI and U2 RNAs, respectively. The pol II

product of U2/7SK is 63 nt ,while the pol III product is 57 nt.

Primer extension analysis was carried out as described by

Murphy et al. (1992) using the oligo described therein for the

U2/7SK RNAs and an oligo of the sequence: 5V-CAGATAC-TACACTTGATCCTCTAGAGC-3Vfor the U2 maxi RNAs.

All constructs were prepared by PCR.

Fig. 1. The whole promoter of the U2 gene is bound by proteins in

unsynchronised HeLa cells. (A) The structure of the U2 promoter is shown

with the relative positions of the DSE and PSE and the U25Vprimer used for

linker-mediated PCR. (B) Methylation protection on the nontranscribed

strand of the U2 promoter. Naked DNA (nDNA) and cellular DNA (vDNA)

was treated with DMS and prepared as described in Section 2.1. In this and

subsequent figures, promoter elements are shown at the left of the figure, and

numbers represent the nucleotide position relative to the start site of trans-

cription. In this and subsequent figures, protected G residues (empty arrows)

are shown to the right of the figure. (C) Protection from DNase I digestion on

the nontranscribed strand of the U2 promoter. Naked DNA (nDNA) and

cellular DNA (vDNA) was treated with DNase I and prepared as described in

Section 2.1. In this and subsequent figures, protected regions are indicated by

empty boxes to the right of the figure and filled arrows at the right indicate

hypersensitive sites. (D) Protection from digestion by micrococcal nuclease

on the nontranscribed strand of the U2 promoter. Naked DNA (nDNA) and

cellular DNA (vDNA)was treatedwithmicrococcal nuclease and prepared as

described in Section 2.1. In this and subsequent figures, boxes bordered by

dashed lines indicate partial protection.

3. Results

3.1. The whole promoter of the U2 gene is bound by

proteins in unsynchronised HeLa cells

The human U2 snRNA genes are located together on

chromosome 17 in arrays of highly conserved 6 kilobase (kb)

tandem repeats, and the number of repeats varies from 6 to

more than 30 (Pavelitz et al., 1995). The DSE of these genes

contains four potential factor binding sites: three Sp1 sites

and an octamer motif, which are all protected from methyl-

ation and DNase I digestion in the presence of nuclear extract

(Janson et al., 1987). Sp1 and Oct-1 bind cooperatively to

these sites in vitro and the function of the elements is

interdependent in vivo although full enhancer activity

requires only one Sp1 site (Janson and Pettersson, 1990).

PTF interacts specifically with the U2 PSE in gel retardation

(Murphy et al., 1992) and DNase I footprinting studies

(D.C.B. and S.M. unpublished observations). In order to

determine which of these sites interacts with proteins in the

living cell, in vivo footprinting was carried out.

Fig. 1 shows the results of linker-mediated PCR over the

nontranscribed strand of the DSE of the U2 gene. A

schematic diagram of the promoter of the U2 snRNA gene

and the primers used for linker-mediated PCR is shown in

Fig. 1A. Primers were designed complementary to sequen-

ces of the GenBank U57614 and L37793 (Pavelitz et al.,

1995) U2 repeats. There are only a few differences between

these two repeats upstream of the coding region, and it is

likely that these primers will also detect several other U2

genes. Cells were treated with DMS, which methylates the

N7 of accessible G residues in the major groove of the DNA

helix (Fig. 1B) and nuclei were treated with DNase I that

creates single-strand breaks in the minor groove (Fig. 1C) or

micrococcal nuclease (Fig. 1D) that introduces double-

strand cuts preferentially in linker DNA between nucleo-

somes (Zhao et al., 2001) (Materials and methods).

By comparison with DNA treated to remove all interact-

ing proteins (nDNA), extensive protection from methylation

and from DNase I digestion is evident over all three Sp1

sites and the Oct-1 binding site within the DSE in vivo

(vDNA). All detectable guanine residues within these ele-

ments are highly protected, and C-251, just upstream of

Sp12 is the only residue between positions �261 and �211

unprotected from DNase I digestion. Schaub et al. (1997)

noted a potential binding site for the transcription factor Staf

between �201 and �183 just downstream of the DSE.

However, no protection from methylation is evident in this

region. The pattern of DMS and DNase I protection over the

DSE of the endogenous U2 genes is very similar to that seen

after incubating U2 constructs with nuclear extract in vitro

(discussed in Section 4.1).

D.C. Boyd et al. / Gene 315 (2003) 103–112106

There is also protection against methylation and DNase

I digestion over the PSE (Fig. 1B and C) (see also Fig. 2)

and regular DNase I hypersensitive sites between the DSE

and PSE (Fig. 1C). Cleavage by micrococcal nuclease is

reduced over the whole promoter from the DSE to +14

downstream from the transcription start site with hypersen-

sitive sites around +15/+17. Protection over the PSE and

downstream is particularly apparent (Fig. 1D), suggesting

extensive and stable binding of factors near the transcrip-

tion start site. The DNase I hypersensitive sites at approx-

imately 10–11 base pair (bp) intervals and protection from

micrococcal nuclease digestion between the DSE and PSE

suggests that a nucleosome is positioned on at least some

of the U2 promoters, and the micrococcal nuclease cleav-

age site at �204 just downstream from the DSE may mark

the upstream boundary.

Fig. 2. The DNA between the PSE and the start site of transcription of the

U2 gene is protected from methylation and digestion. (A) The position of

the U2 PSE primer is shown relative to the U2 promoter. (B) Methylation

protection on the nontranscribed strand of the U2 promoter. (C) Protection

from DNase I digestion on the nontranscribed strand of the U2 promoter. In

this and subsequent figures, grey arrows at the right indicate relatively weak

hypersensitive sites, and the size of the arrow correlates to the relative

degree of hypersensitivity. (D) Protection from digestion by micrococcal

nuclease on the nontranscribed strand of the U2 promoter.

The high level of protection suggests that most, if not all,

copies of the U2 gene detected here are bound by factors in

the majority of the cells used for this analysis. Thus, the

promoters of all genes within the cluster may be occupied

by factors in all cells during interphase.

3.2. The DNA between the PSE and the start site of

transcription of the U2 gene is protected from methylation

and digestion

PTF binds to the essential PSE located between positions

�60 and �43 of the U2 genes (Murphy et al., 1992) and is

required for transcription of the pol II-transcribed U1 snRNA

gene in vitro (Yoon et al., 1995). Although there is no TATA

box in their promoters, transcription of pol II-dependent

snRNA genes requires the TATA-binding factor TBP

(reviewed by Hernandez, 2001). Insertion of a TATA box

effectively abolishes transcription by pol II, and instead, pol

III is recruited (Lobo and Hernandez, 1989). TBP is also

required for transcription of the pol III-dependent snRNA

genes and binds directly to the TATA box in these genes

(Lobo et al., 1991; Boyd et al., 1995). Therefore, how TBP

interacts with the promoter is critical for differential poly-

merase selection. The results of micrococcal nuclease diges-

tion shown in Fig. 1D indicate that factors interact with DNA

downstream of the PSE in the U2 gene. In order to analyse

this region in more detail, linker-mediated PCR was carried

out using primers upstream (nontranscribed strand) (Fig. 2)

and downstream (transcribed strand) (Fig. 3) of the proximal

promoter region of the U2 gene. The sequenced U2 genes are

highly conserved in this region, and these primers are likely

also to detect some, if not all, of the other genes in the cluster.

Analysis was carried out on DNA samples from DMS-

treated cells (Figs. 2B and 3B), and DNase I- or micrococcal

nuclease-treated nuclei (Figs. 2C, D and 3C).

On the nontranscribed strand of the PSE (Fig. 2B) four

residues within the PSE are protected from methylation (and

see also Fig. 6B). Only the G at �56 within the PSE is not

protected. There are also two strongly protected G residues

between the PSE and the start site of transcription at

positions G-28 and G-22, and a hypersensitive site at G-

19. This result suggests that a protein interacts with the

major groove of DNA between the PSE and the start site of

the U2 gene, although this region has not previously been

implicated in transcription of the U2 gene.

The DNase I digestion pattern (Fig. 2C) indicates that

there is an extended DNase I footprint over the PSE of the

U2 gene, the 5V and 3V borders of which are located at

positions �83 and �36, respectively, with hypersensitive

sites at either end of the footprint at C-84 and C-34. There is

also some protection from digestion by DNase I between

�22 and �30 and a hypersensitive site downstream of this

region (Fig. 2B) (see also Fig. 1C). As seen in Fig. 1D, the

whole region between the PSE and the start site is protected

from digestion by micrococcal nuclease, with hypersensitive

sites around +15/+17 (Fig. 2D).

Fig. 3. Protection from methylation and digestion on the transcribed strand

of the U2 gene. (A) The position of the U23Vprimer is shown relative to the

U2 promoter. (B) Methylation protection on the transcribed strand of the U2

promoter. (C) Protection from DNase I digestion on the transcribed strand

of the U2 promoter.

D.C. Boyd et al. / Gene 315 (2003) 103–112 107

There is little protection against methylation on the

transcribed strand of the PSE with the G residue at �44

obviously protected (Fig. 3B). In addition, the G residues at

�53 and �41 appear to be hypersensitive. There are also

slightly hypersensitive G residues at �34 and �36 and

some protection of G-32 and -26 between the PSE and the

transcription start site, again suggesting that a protein

interacts with the DNA here.

On the transcribed strand, there is a highly extended

DNase I footprint over the PSE and upstream (Fig. 3C),

presumably due, at least in part, to binding of PTF. The

downstream border of the footprint is around the A at �35.

Downstream of the PSE are a number of DNase I hyper-

sensitive sites at positions A-29, G-27, G-18, G-16, C-15,

G-14 and G-8. There are also regular hypersensitive sites

between the PSE and DSE at �121, �124, �132/134,

�145, �166, �174 and �193/194.

These results suggest that PTF interacts primarily with

the major groove of the nontranscribed strand of the U2 PSE

and that either PTF or another factor(s) interacts with the

major groove of both strands downstream of the PSE. The

presence of DNase I hypersensitive sites suggests that this

region of the DNA is distorted in vivo. In addition, the

regular DNase I hypersensitive sites between the PSE and

DSE support the notion that a nucleosome is positioned

between the two elements.

3.3. Mutation of the region between �33 and �20 affects

initiation and polymerase specificity

Protection from both methylation and DNase I digestion

between positions �32 and �20, flanked by DMS hyper-

sensitive sites at �19 and �36, (summarised in Fig. 4A)

suggests that a factor(s) interacts with the major groove of

the DNA between �32 and �20. Previous studies indicate

that there are no strict sequence requirements downstream

of the PSE of the U2 gene, although mutation of sequences

between �33 and �42 and between �20 and �11 can

affect transcription efficiency (Hernandez and Lucito,

1988), and sequences around the start site of transcription

appear to play a role in initiation (Hernandez and Lucito,

1988; Lobo et al., 1990). Although mutation of the

sequence between �14 and �37 had little effect in a

linker-scanning analysis of the U2 gene (Hernandez and

Lucito, 1988), the GC-rich region between �33 and �20 is

quite similar in both U1 and U2 genes (see Fig. 4B),

suggesting that this sequence is not entirely neutral. We

have therefore mutated every bp between �33 and �20 in

a U2 maxigene (Murphy, 1997) (U1 homology knockout,

U1HKO, Fig. 4B) to destroy any homology to the same

region of the U1 gene and to reduce the GC content. The

effect on transcription was analysed using an S1 nuclease

assay as previously described (Murphy, 1997) (Fig. 4C).

The pol III-transcribed VAI gene was used as an a-

amanitin resistant, co-transfection control. In agreement

with the findings of Hernandez and Lucito (1988), the

14-bp mutation has little effect on the efficiency of tran-

scription (compare lanes 1 and 3). However, the mutation

appears to affect initiation since an extra, larger S1 product

is visible in lane 3. Primer extension analysis (Fig. 4D)

confirmed that a small amount of initiation occurs at �2 on

the mutated template (compare lanes 1 and 2) (see Fig. 4E).

This result suggests that interaction of factors with sequen-

ces downstream of the PSE is involved in setting the site of

Fig. 5. Mutation of the region between �33 and �20 affects polymerase

specificity. (A) The sequence of the U2/7SK-TA mutant between �13 and

�40 is shown below the wild-type U2 sequence. The bases conserved in the

U2 and U1 genes and the mutations made in U2/7SK-TA are marked in bold.

(B) S1 mapping of transcripts from U2/7SK constructs. Here and in (C), the

expected position of products from pol II- or pol III-transcribed RNA and

VAI is indicated at the right and the construct transfected is indicated above

the lanes. (C) Primer extension of transcripts from U2/7SK constructs. (D)

The 5V ends of the primer extension products are indicated above the

sequence of the +1 region of the U2/7SK constructs by filled arrows. The

size of the arrows corresponds to the relative strength of the products.

Fig. 4. Mutation of the region between �33 and �20 affects initiation. (A)

A summary of the results of DMS and DNase I footprinting of the region

downstream of the U2 PSE. Protection from DNase I is indicated by boxes.

Broken lines indicate that the protection is relatively weak. G residues

protected from methylation are shown in bold type. DNase I hyper-

sensitivity is indicated by hatched arrows and DMS hypersensitivity by

filled arrows. The size of the arrows corresponds to the relative strength of

hypersensitivity. (B) The homology between the U2 and U1 genes between

�20 and �33 is indicated in bold and boxed. The base changes made in

U1HKO are indicated in bold below. (C) S1 mapping of transcripts from the

wild-type U2 maxigene (WTU2, lanes 1 and 2) and U1HKO maxigene

(lanes 3 and 4). Here and in Fig. 5, the addition of a-amanitin to the cells

after transfection is indicated above the lanes. VAI was used as a co-

transfection control. The position of the products after S1 digestion is

indicated at the right. (D) Primer extension of transcripts from WTU2 and

U1HKO maxigenes. The expected position of the primer extension product

of a properly initiated transcript is noted as +1 at the right. The shorter

primer extension bands indicated with an asterisk most likely reflect the

effect of strong secondary structure in the template on reverse transcriptase.

(E) The 5Vends of the primer extension products are indicated above the

sequence of the WTU2 and U1HKO +1 region by filled arrows. The size of

the arrows corresponds to the relative strength of the products.

D.C. Boyd et al. / Gene 315 (2003) 103–112108

initiation. The addition of a low level of a-amanitin to the

transfected cells inhibited transcription from both the wild-

type U2 and U1HKO templates, confirming that they are

transcribed by pol II (Fig. 4C, lanes 2 and 4).

It is unlikely that any pol III-synthesised RNA would be

detected using these constructs since a strong pol III

transcription termination sequence, GCCTTTTGGC, locat-

ed between +12 and +21 of the wild-type U2 gene is

retained in the maxigene. In addition, any pol III-transcribed

RNA will not contain the signals for capping and may be

unstable (Shumyatsky et al., 1993). Thus, any effect of the

U1HKO mutation on polymerase specificity of the U2

promoter may not be detected. To remove the pol III

terminator and to effectively stabilise any pol III transcripts

initiated from these constructs, we fused the region from

�400 to +6 of the wild-type U2 and U1HKO constructs to

the coding region of the pol III-transcribed 7SK gene

(WTU2/7SK and U2/7SK-U1HKO, see Fig. 5D). This

ensures that any PSE-dependent transcription initiated by

pol III at the right distance from the PSE will result in full-

length, capped 7SK RNA. As a control for the system, we

also introduced a 9-bp TATA box into the WTU2/7SK

construct to give the same sequence that Lobo and Hernan-

dez (1989) found converted the U2 gene to a pol III snRNA

gene type (U2/7SK-TA, see Fig. 5A). A marked 7SK gene

was also transfected. The RNA from these transfected

constructs was assayed by S1 nuclease and primer extension

analysis (Fig. 5B–D). Transcripts from WTU2/7SK are

almost exclusively synthesised by pol II as determined by

sensitivity to low levels of a-amanitin (Fig. 5B, lanes 1, 2

and Fig. 5C, lanes 1, 2), indicating that sequences in the

7SK coding region do not affect polymerase specificity.

These transcripts initiate at or very close to the expected

position in the U2 sequences (Fig. 5C and D). However, the

U1HKO mutation results in detectable transcription by pol

III starting at the 7SK+1 position (Fig. 5B, lanes 3–5C,

lanes 3–5D). Thus, replacement of the region between �33

and �20 with a mutated sequence that is less GC rich allows

some transcription by pol III to occur in the absence of a

recognisable TATA box at �25. Introduction of the TATA

box into the WTU2/7SK construct causes a dramatic in-

crease in the amount of transcription by pol III, although

D.C. Boyd et al. / Gene 315 (2003) 103–112 109

there is still a small amount of transcription by pol II (Fig.

5B–6 and Fig. 5C–6). In contrast, the 7SK gene appears to

be exclusively transcribed by pol III Fig. 5B(, lanes 7, 8 and

Fig. 5C, lane 7).

These results suggest that the sequence between �33 and

�20 contributes to accurate positioning of the start site and

maintaining the polymerase specificity of the U2 gene.

3.4. Transcription factors are released from the U2

promoter in mitosis

In an unsynchronised population of HeLa cells, the

majority of the U2 genes are likely to be actively transcribed.

However, during mitosis, transcription is globally extin-

guished (reviewed by John and Workman, 1998). To deter-

mine the fate of factors bound to the U2 promoter during

mitosis, we have carried out in vivo footprinting on a pop-

ulation highly enriched for mitotic cells using the shake off

method (see Materials and methods) (Fig. 6). Fig. 6A shows

the results of footprinting the DSE region of the nontran-

Fig. 6. Transcription factors are lost from the U2 promoter in mitosis. (A)

Methylation protection of the nontranscribed strand of the DSE in

unsynchronised (vDNA) and mitotic-enriched (mitotic vDNA) cells using

the U25Vprimer. (B) Methylation protection of the nontranscribed strand of

the PSE in unsynchronised (vDNA) and mitotic-enriched (mitotic vDNA)

cells, using the U2PSE primer. Symbols are as in Figs. 1 and 2.

scribed strand, and Fig. 6B shows the results of footprinting

the PSE and downstream region on the same strand. The level

of footprinting over the DSE is drastically reduced in the

mitotically enriched cells (cf. Fig. 6A vDNA and mitotic

vDNA). However, the pattern is not identical to that seen in

the nDNA lane, and a low level of binding to the Sp1 sites is

still detectable. In contrast, the G in the Oct-1 binding site is

no longer protected and has become slightly hypersensitive.

Similarly, the degree of footprinting/hypersensitivity over the

PSE region is markedly reduced but not abolished. Little

residual footprinting/hypersensitivity is detected over the

region downstream of the PSE, although it should be noted

that protection of G residues in the PSE and downstream was

reduced in this sample of unsynchronised cells (vDNA)

compared to the sample used for Fig. 1, possibly due to

differences in the population of cells used. These results

suggest that transcription of the U2 genes is downregulated at

mitosis through loss of factors from the template.

4. Discussion

The human snRNA genes transcribed by pol II have

specialised TATA-less promoters containing only one es-

sential promoter element, the PSE, and a transcriptional

enhancer, the DSE. Several of the basic transcription factors

required for preinitiation complex formation on mRNA

genes are also required for transcription of these snRNA

genes, and the factor-binding sites comprising DSEs are also

found in mRNA genes. Interestingly, insertion of a TATA

box at �25 of this type of promoter causes recruitment of

pol III (Lobo and Hernandez, 1989), and mutation of the

TATA box at �25 in the highly related, PSE-containing

promoter of pol III-dependent snRNA genes causes recruit-

ment of pol II (Boyd et al., 1995). Thus, the combination of

PSE and TATA favours the formation of a pol III-specific

preinitiation complex, whereas pol II-specific interactions

predominate in the absence of a TATA box. The PSE-

binding factor PTF/PBP/SNAPc functions on snRNA genes

transcribed by both pol II and pol III (reviewed by Hernan-

dez, 2001). Since the TATA-binding protein TBP is also

required for transcription of both types of snRNA gene, the

primary basis for differential polymerase recruitment

remains unclear. The precise architecture of factors bound

to snRNA gene promoters must therefore determine both the

transcriptional activity and the polymerase specificity of

these highly expressed genes.

4.1. In vivo protection over the DSE of the U2 gene is very

similar to that seen in vitro

The octamer motif and three Sp1 binding sites of the U2

DSE are footprinted by proteins in nuclear extract in vitro,

and oligos containing Oct-1 or Sp1 binding sites compete

away the footprint (Janson et al., 1987). Interestingly, the in

vivo pattern of protection from DMS and DNase I over the

D.C. Boyd et al. / Gene 315 (2003) 103–112110

DSE is very similar to that seen in vitro. Although different

genes were analysed, the sequences over the DSE and

downstream region are highly homologous. The only dif-

ference in the pattern of DMS protection is that one G

residue within Sp12 is protected in vivo and unprotected in

vitro. DNase I footprinting shows two changes: residues

around �250 are hypersensitive in vitro and merely unpro-

tected in vivo, and the hypersensitive site 3Vof the DSE is

slightly further downstream in vivo than in vitro. These

results indicate that Sp1 and Oct-1 bind to these sites in vivo

as expected and suggest that the chromosomal context

affects the pattern of DNase I hypersensitive sites. Activa-

tion of transiently transfected U2 constructs requires only

one Sp1 site (Janson and Pettersson, 1990), but all three

binding sites of endogenous U2 genes are protected in HeLa

cells. This may indicate that Sp1 is not limiting in these cells

and binds to any available site even when not required for

high levels of activation. Alternatively, occupation of all

sites may be required for high levels of activation in the

chromosomal context.

4.2. Is a nucleosome positioned between the DSE and PSE

of all snRNA genes?

Several lines of evidence suggest that a nucleosome lies

between the DSE and PSE of snRNA genes. The distance

between the DSE and PSE of mammalian snRNA genes is

remarkably constant (f150 bp) and corresponds to the

distance required for binding of a histone octamer to the

DNA. In vitro, potentiation of PTF binding by Oct-1, and

transcription of the 7SK gene are inefficient unless the DSE

and PSE are close to one another (Murphy et al., 1992),

indicating that there may be a mechanism juxtaposing these

elements in vivo. Furthermore, positioning of a nucleosome

between the DSE and the PSE of the human U6 gene

increases transcription in vitro (Stunkel et al., 1997; Zhao

et al., 2001), and in vivo footprinting studies on the 7SK,

U6 and U1 genes suggest that a nucleosome is positioned

between the DSE and PSE of these genes in living cells

(Boyd et al., 2000; Zhao et al., 2001). When DNA is

wrapped around a histone octamer, DNase I will only cut

at positions where the minor groove is facing outwards,

which occurs at 10–11-bp intervals (discussed by Boyd et

al., 2000). A number of regular DNase I hypersensitive sites

occur approximately 10 bp apart on both strands of the U2

promoter between the DSE and PSE. In addition, digestion

by micrococcal nuclease is reduced in this region as

expected for nucleosomal DNA. We therefore favour the

hypothesis that a nucleosome is also positioned between the

DSE and PSE of the U2 genes.

4.3. Different residues are protected within the PSEs of the

7SK and U2 genes in vivo

The protection observed in vivo over the PSE of the U2

is most likely due largely to PTF binding to the DNA, since

there is strong in vitro evidence that binding of this factor to

the PSE is required for transcription. Interestingly, the in

vivo DMS footprint over the PSE of the U2 gene is different

from that on the PSE of the pol III-transcribed 7SK gene

(Boyd et al., 2000). All G residues in the 7SK PSE are

protected from methylation apart from the G at �62 on the

nontranscribed strand, while three out of the four G residues

in the upper strand of the U2 PSE are unprotected. Different

G residues are protected within the conserved 3V-ANTGG-5Vof the PSE in the U2 and 7SK genes (see Murphy et al.,

1989, 1992), indicating that PTF makes different major

groove interactions with the two promoters. This may be

due to differences in the sequences of the two PSEs (see

Murphy et al., 1989, 1992), and/or to the effects of other

factors (e.g., enhancer binding proteins or TBP) on the

binding of PTF. For instance, direct interaction between

TBP and the TATA box of the pol III-dependent 7SK gene

may affect the interaction of PTF with the PSE. In addition,

differerences in PSE sequences may favour either pol II- or

pol III-specific interactions. In Drosophila melanogaster

snRNA genes, as little as three nucleotide (nt) difference

between PSEs can affect both binding of transcription

factors and polymerase selection (Jensen et al., 1998).

4.4. Factors interact with sequences downstream from the

U2 PSE in vivo

G residues between �32 and �22 are protected from

methylation, and there are hypersensitive sites at �19 and

�34. In addition, the nontranscribed strand is protected

from digestion by both DNase I and micrococcal nuclease

downstream of the PSE. The DNA around this region also

appears to be distorted, as shown by DNase I hypersensi-

tive sites predominantly on the transcribed strand. These

findings suggest that a factor(s) interact(s) stably with the

major groove of the DNA in this region and cause(s)

perturbations in the structure of the DNA. Although

mutation of the nucleotides protected from methylation

does not cause a reduction in transcription (Fig. 4 and

Hernandez and Lucito, 1988), both initiation and polymer-

ase specificity are affected, highlighting the importance of

the interaction detected.

Since PTF alone does not protect the region downstream

of the U2 PSE from methylation in vitro (D.C.B. and S.M.,

unpublished observations) it is likely that another factor is

binding here. Recently, it has been shown that TFIIB has

some sequence binding preferences (Lagrange et al., 1997),

and the sequence between �25 and �31 of the U2

promoter, 5V-GGACGGT-3V is similar to the GC-rich con-

sensus TFIIB recognition element (BRE) 5V-G/CG/CG/ACGCC-3V. In addition, TFIIB plays a major role in start

site selection (Lagrange et al., 1997). Thus, we are tempted

to speculate that TFIIB contributes to the footprint down-

stream of the PSE on the U2 gene and that initiation and

polymerase specificity are affected when its interaction with

the DNA is altered.

Fig. 7. An ‘‘exclusion’’ model for polymerase specificity of snRNA genes.

In the absence of TBP binding downstream of the PSE, a pol II-specific

factor gains access to the DNA between �20 and �33 and binds with little

sequence specificity, stabilised by protein–protein interactions for instance

with PTF. Pol III-specific factors, on the other hand, can only interact stably

when TBP is bound to the TATA box. In addition, interaction of PTF with

the PSE may be different on pol II- and pol III-transcribed genes.

D.C. Boyd et al. / Gene 315 (2003) 103–112 111

A simple ‘‘exclusion’’ model for polymerase specificity

is consistent with our findings, if we consider that the

factor binding downstream of the PSE is likely to be

specific for pol II (Fig. 7). In the absence of a binding

site for TBP, a factor critical for formation of a pol II-

specific preinitiation complex (e.g., IIB) can bind to the

�20/�33 region. The sequence similarity in this region of

the U1 and U2 snRNA genes suggests that it contributes to

the efficient expression of these highly transcribed genes.

However, the sequence of this region is not conserved in

all pol II-transcribed snRNA genes and non-sequence-

specific binding of factors may be favoured by GC-rich

sequences and/or promoted and stabilized by protein–

protein interactions, for instance with PTF. When a TATA

box is present, access to the template is blocked by TBP

binding directly to the promoter and the pol II-specific

factor is excluded, thereby ensuring that pol II is not

recruited. A pol III-specific factor, instead, is recruited

only when TBP is bound directly to the TATA box. In

support of this model, we have shown that the pol III- and

snRNA-specific IIB homologue TFIIIB50/BRFU interacts

strongly with template DNA only when TBP is bound to

the TATA box (Cabart and Murphy, 2001). As discussed

above, the precise interaction of PTF with the PSE may

also be affected by sequence differences and differential

downstream protein/protein interactions. In this model, pol

II recruitment is the default state, but the exact sequence of

the PSE and the presence/absence of a BRE can influence

the final pol II versus pol III outcome.

4.5. Transcription factors are lost from the U2 promoter in

mitosis

At mitosis, transcription is globally extinguished, and

transcription factors can undergo mitosis-specific modifi-

cation that reduces the ability to interact with promoters

(reviewed by John and Workman, 1998). Since U2 RNA is

needed at high levels in all cells, the U2 genes are good

candidates for ‘‘mitotic bookmarking,’’ where factors

bound to the promoter during mitosis ensure immediate

expression of the genes on exit from mitosis (reviewed by

John and Workman, 1998). However, it appears that most

of the factors bound to the U2 promoters during interphase

are displaced at mitosis and no new major groove contacts

are apparent. Oct-1 undergoes mitotic phosphorylation that

decreases its ability to bind to DNA (Segil et al., 1991),

and the binding activity of Sp1 is reduced in mitotic

extracts (Martinez-Balbas et al., 1995), which explains

the loss of these factors from the U2 DSE. The loss of

Oct-1 from the DSE may in turn lead to the loss of PTF

since Oct-1 potentiates binding of PTF to the PSE (Murphy

et al., 1992). Contamination of the cell sample with non-

mitotic cells is unlikely to account entirely for the residual

footprinting detected over the DSE and PSE since the Oct-

1 site in the DSE is no longer protected and the pattern of

protection over the DSE has changed rather than simply

decreased. Since there are multiple copies of the U2 genes,

the residual footprinting detected over the DSE and PSE

may indicate that a subset of genes retains promoter-bound

factors in each cell. However, we think it more likely that

some factors are left behind on some genes by chance

rather than by design since the single-copy pol III-tran-

scribed 7SK gene has approximately the same proportion

of residual footprinting over the PSE at mitosis (D. C. B.

and S. M., unpublished observations). Mitotic Sp1 still

binds detectably to DNA and the SP1 sites in the promoter

of the hsp70 gene are also still partially protected in mitosis

(Martinez-Balbas et al., 1995). Thus, the residual foot-

printing on the U2 genes in mitosis (Fig. 6) may simply

reflect a general low level of retention of some factors in

condensed chromatin.

Acknowledgements

We would like to thank Ingo Greger for help with in vivo

footprinting, Patricia Uguen and Nicolas Buisine for help

with the figures and Patricia Uguen and Joanne Medlin for

critically reading the manuscript. D.C.B. and S.M. were

supported by MRC Senior Fellowship No. G117/309, and

A.P. by a Royal Society Dorothy Hodgkin Fellowship.

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