The EMBO Journal vol.10 no.10 pp.3007-3014, 1991

POU proteins bend DNA via the POU-specific domain

C.Peter Verrijzer, Joost A.W.M.van Oosterhout, Willem W.van Weperen and Peter C.van der Viiet Laboratory for Physiological Chemistry, University of Utrecht, Vondellaan 24a, 3521 GG Utrecht, The Netherlands Communicated by H.S.Jansz

POU proteins constitute a family of ubiquitous as well as cell type-specific transcription factors that share the conserved POU DNA binding domain. This domain consists of two distinct subdomains, a POU-specific domain and a POU homeodomain, that are both required for high affinity sequence-specific DNA binding. In a circular permutation assay, several POU proteins, including Oct-i, Oct-2A, Oct-6 and Pit-1, demonstrated a position dependent mobility of the protein-DNA complexes, suggesting induction of DNA bending. This was confirmed by detection of relative bend direction, using pre-bent DNA, and by enhanced ligase mediated cyclization. Bending was caused by interaction with the POU domain. By contrast, binding of the POU homeodomain did not distort the DNA structure, indicating that the POU-specific domain confers DNA bending. Key words: DNA bending/DNA replication/octamer sequence/POU domain/transcription factors

Introduction During initiation of DNA replication, recombination, or initiation of transcription, large nucleoprotein complexes are formed, in which multiple protein-DNA and protein -protein interactions are involved. In these complexes the DNA is frequently distorted from its regular, linear, double helical structure. A well described and experimentally accessible mode of distortion is DNA bending. Bending can be intrinsic to a specific DNA sequence or can be imposed as a consequence of binding of a particular protein. Induction of DNA bending has been observed for several prokaryotic proteins such as the Escherichia coli CAP protein (Wu and Crothers, 1984), IHF (Prentki et al., 1987; Stenzel et al., 1987), the lambda 0 protein (Zahn and Blattner, 1985), 429 protein p4 (Rojo et al., 1990) and E. coli RNA polymerase (Heumann et al., 1988) as well as for some eukaryotic proteins involved in transcriptional control, for example Drosophila heat-shock transcription factor (Shuey and Parker, 1986), Xenopus TFIIIA (Schroth et al., 1989) and yeast mitochondrial RNA polymerase (Schinkel et al., 1988). Synthetic curved DNA can functionally replace IHF or CAP induced bending, demonstrating that bending is not a mere side effect of DNA -protein complex formation, but can be of functional Oxford University Press

significance in biological processes (Snyder et al., 1989; Goodman and Nash, 1989; Bracco et al., 1989). The POU domain is an evolutionarily conserved motif present in a family of transcription factors (for reviews, see Herr et al., 1988; Ruvkun and Finney, 1991). The POU protein family includes tissue-specific transcription factors such as Pit- 1, which regulates transcription of prolactin and growth hormone genes (Ingraham et al., 1988; Bodner et al., 1988; for review, see Karin et al., 1990) and Oct-2, which is involved in immunoglobulin expression (Clerc et al., 1988; Miiller-Immergliick et al., 1988; Scheidereit et al., 1988; Ko et al., 1988). Others have been implicated in developmental regulation e.g. Oct-3/4 (Okamoto et al., 1990; Rosner et al., 1990; Scholer et al., 1990) Oct-6 (Meijer et al., 1990; Monuki et al., 1990; Suzuki et al., 1990; He et al., 1991) or the Caenorhabditis elegans Unc-86 gene product (Finney et al., 1988). Oct-I is ubiquitously expressed and activates transcription of housekeeping genes such as the histone H2B gene and snRNA genes (Sturm et al., 1988). Furthermore, it enhances initiation of adenovirus (Ad) DNA replication (Verrijzer et al., 1990a). Within the POU domain, two conserved regions can be recognized: a 67-74 amino acids long POU-specific (POUs) domain and a 60 amino acids long POU homeodomain (POUHD), separated by a short non-conserved sequence. Whereas the POUHD is distantly related to the classic homeodomain proteins, the POUs domain is unique to the POU protein family. The homeodomain was first recognized in proteins encoded by the homeotic genes and several other genes that play a central role in eukaryotic development (for reviews, see Scott et al., 1989; Affolter et al., 1990). In these proteins, the homeodomain represents the complete DNA binding region (Hall and Johnson, 1987; Mihara and Kaiser, 1988; Muller et al., 1988). Recent structural analysis of homeodomains of the Drosophila Antennapedia and Engrailed proteins revealed that they contain three well defined a-helices, two of which form a helix-turn-helix motif similar to that observed in various prokaryotic repressors (Qian et al., 1989; Kissinger et al., 1990). In contrast to the classic homeodomain proteins, DNA binding of the POU domain family requires the POUs domain in addition to the POUHD (Sturm and Herr, 1988; Ingraham et al., 1990; Verrijzer et al., 1990a,b). The POUs domain contributes to both binding affinity and specificity via direct DNA contacts (Verrijzer et al., 1990b; Ingraham et al., 1990; Kristie and Sharp, 1990). The POUs domain is also required for enhancement of initiation of adenovirus DNA replication. No additional protein regions outside the POU domain are required for this function, demonstrating a direct biological function of this DNA binding domain (Verrijzer et al., 1990a). In order to delineate further the interaction between the POU domain and DNA and to study the specific function of the POUs domain, we investigated whether binding of

3007

C.P.Verrijzer et al.

the POU domain induces changes in the DNA structure. We report here that the POUs domain is required for bending of the octamer recognition sequence and we discuss the possible consequences for the control of transcription and DNA replication by POU proteins.

Results POU proteins induce DNA bending Bent DNA can be detected by its anomalous migration in polyacrylamide gels (Wu and Crothers, 1984). The position of the bend determines the electrophoretic mobility. Anomalous migration is maximized when the bend is located in the centre of a fragment and minimized when it is located near the end. We cloned the canonical octamer sequence (ATGCAAAT) from the Ad4 origin of DNA replication into the circular permutation vector pBend2 (Kim et al., 1989; Zwieb et al., 1989). Digestion of the resulting plasmid, pOctaBend, with a number of restriction endonucleases (Figure 1) generates fragments of identical size (143 bp) and base composition, but with variant positions of the octamer. Figure 2A shows the electrophoretic mobility of circularly permutated fragments incubated with purified Oct-I protein. No anomalous migration is observed for the free DNA, demonstrating the absence of any intrinsic curvature. By contrast, migration of the Oct-I DNA complex is clearly dependent on the position of the octamer within the fragment. These results indicate that binding of Oct-I induces DNA bending. To investigate if induction of DNA bending is a property shared by other members of the POU protein family, we also tested several other proteins containing a POU domain. We employed a recombinant vaccinia virus system to express cDNAs encoding Oct-2A (Muiller-Immergluick et al., 1988), Oct-6 (Meijer et al., 1990) and a chimeric Oct-I protein in which the POU domain has been replaced by the Pit- I POU domain (Stern et al., 1989). The Oct-2A POU domain is closely related to that of Oct-1, but Oct-6 and Pit-I are more diverged and belong to different POU classes (He et al., 1989). The electrophoretic mobility of DNA fragments with the octamer site located either in the centre (D) or terminally (A and H) was monitored after binding of the indicated proteins (Figure 2B). For all POU proteins, the mobility of the protein -DNA complex was strongly dependent on the position of the octamer within the fragment. These results indicate that DNA bending is a common feature of POUdomain containing proteins. The isolated Oct-I POU domain also shows a position dependent mobility (Figure 2B, lanes 13-15). The differences in relative mobility were larger for the intact proteins than for the POU domain, suggesting that intact Oct-I induces stronger DNA bending than the POU domain. We analysed a number of N-terminal and C-terminal deletion mutants of Oct-I (Verrijzer et al., 1990a) to investigate whether we could find a specific region outside the POU domain responsible for this difference, but none was found (not shown). As discussed below, the reason for this discrepancy most likely is the difference in shape between intact Oct-I and the POU domain. Detection of the relative bend direction

Using isomers with varied helical phasing between two DNA bends, Zinkel and Crothers (1987) introduced an elegant 3008

Ad4

GCCTTATTTGCATATT CGGAATAAACGTATAA

Ad2

CCCTCATTATCATATT GGGAGTAATAGTATAA

ICP4 AB RP

ATCTCATTACCGCCCT

TAGAGTAATGGCGGGA

A B

C D

C DE F G H

C.

F.

G.

H

_

Fig. 1. Structure of the restriction fragments used to investigate DNA bending. Oligonucleotides containing the octamer sites from the Ad4 or Ad2 origin, or the herpes simplex virus ICP4 TAATGARAT motif were cloned into the XbaI site of the circular permutation vector pBend2 (Kim et al., 1989), to generate pOctaBend, pAd2Bend and pICP4Bend, respectively. In the resulting constructs, the octamer motifs are flanked by two direct repeats of 116 bp. This allows the generation of 143 bp, circularly permuted fragments (A-H) using the restriction enzymes MluI (A), NheI (B), (E), NruI (F), KpnI (G) or BamHI (H).

XhzoI (C), EcoRV (D), SmaI

approach for determining the relative DNA bend direction. We cloned the octamer sequence in SB 10-20 DNAs (Drak and Crothers, 1991) containing adenine-tract directed, intrinsically bent DNA (Figure 3A). Using a set of six linkers, varying in length from 10 to 20 bp, the helical repeat between the octamer and the sequence-directed bend was varied. If proteins binding to the octamer sequence induce bending, the mobility of the protein-DNA complexes should depend on the linker length, since this determines the relative orientation of the sequence directed and protein induced DNA bend. The mobility of the protein -DNA complexes is minimal in the cis-isomer, when the end-to-end distance is short, and maximal in the trans-isomer. Figure 3B shows that the mobility of the POU -DNA complex varies in a phase dependent manner. The free DNA also shows anomalous migration, which is due to the amplification of inherent bends (Zinkel and Crothers, 1987). When the relative mobilities of the POU -DNA complexes are divided by the relative mobilities of the free DNA and plotted against the linker length, a clear variation with a 10 bp periodicity is observed (Figure 3C). This confirms that the POU domain induces DNA bending. Furthermore, as argued by Zinkel and Crothers (1987), this assay shows that the POU-induced bend is not a point of induced flexibility but is directed in space. Position dependent differences in electrophoretic mobility of protein -DNA complexes can result from an aberrant shape of the bound protein without DNA bending, as shown for GCN4 (Gartenberg et al., 1990). Since the position of the octamer within the DNA fragments remains virtually identical, the detection of relative bent direction circumvents this problem. The relative mobilities of the pre-bent DNA bound to intact POU proteins were almost identical to those of the POU -DNA complexes (not shown). This indicates that the discrepancy in the circular permutation assay between Oct-I and the POU domain (Figure 2) arises mainly

~

A.

POU proteins bend DNA

S.,

-

Fig. 2. Electrophoretic analysis of circularly permutated POU protein-DNA complexes. (A) Oct-l protein was bound to eight 32P-end labelled 143 bp DNA fragments (A-H) from pOctaBend, described in Figure 1, each with a uniquely positioned octamer motif. Complexes formed were analysed on a 4% polyacrylamide gel. (B) Recombinant vaccinia virus expressed Oct-I (lanes 1-3), Oct-2A (lanes 4-6), Oct-6 (lanes 7-9), chimeric Oct-I/Pit-I fusion protein containing the Pit-I POU domain (lanes 10-12) and the isolated Oct-I POU domain (lanes 13-15) were incubated with DNA fragments in which the octamer motif was located either in the centre (D) or near the ends (A and H) and analysed on a 4% polyacrylamide gel. Protein free DNA is indicated (F).

A. 4

49

1,

"

~~~~~~~~~~~~~° --

1~~~~~~~~~~~~~~~~~~~~~~~

..

~~~~~~~~~~~~~~~~~~~

I

*4 ...

4 .....

- . -. "Z

2 3

A

-

...-.,

7

..

.

Fig. 3. Relative direction of bending assay. (A) Structure of SBocta DNAs. Fragments contain a small bend induced by two GCA6GC tracts, a linker varying in length from 10 to 20 bp, and the canonical octamer sequence (Ad4). The cis and trans isomers are shown. (B) SBocta(IO) (lane 1) to SBocta(20) (lane 6) were bound by the POU domain and analysed by electrophoresis on a 5% acrylamide gel. The number on top of each lane corresponds to the increase in linker length, in bp. (C) The periodicity of variation in POU-DNA complex mobility corresponds to one helical turn. The ratio of the relative mobility for the POU-DNA complex divided by the corresponding free DNA relative mobility is plotted against linker length. Relative mobility is defined as the mobility normalized to the average value for the set of six linker lengths for the POU-DNA complex and the free DNA, respectively.

from differences in structure between intact protein and the POU domain, and not from increased DNA bending by full length Oct-1. The POUs domain is required for DNA bending We studied the effects of degenerate octamer sequences on POU-induced DNA bending. These sites differ considerably

in their affinity for the POU domain, with Kd values ranging from 9x 1011 M for the canonical octamer, present in the Ad4 origin and VH promoter (AATATGCAAATAA), 3.7 x 101 M for the degenerate Ad2 octamer (AATATGATAATGA) and 1.9 x 10-9 M for the TAATGARAT motif from the HSV ICP4 immediate early promoter

(GCGGTAATGAGAT) (Verrijzer et al., 1990b). Despite differences in affinity, all complexes show approximately

3009

C.P.Verrijzer et al.

the same variation in mobility, indicating that the POU domain induces DNA bending in all these sites, irrespective of the binding affinity (Figure 4A). The bending angle ce is defined as the angle by which a segment of the DNA departs from linearity. Although a clear theory for calculating protein-induced bending angles is lacking, an empirical equation LM/LE = cos(a/2) has been formulated (Thompson and Landy, 1988). Here ILM is the mobility of the complex with the protein bound centrally and ,AE is the mobility with the protein bound at an end. Employing this equation we calculated a POU-induced bending angle varying from 290 in a 4% polyacrylamide gel up to 450 in a 10% polyacrylamide gel. For the Ad2 as well as for the TAATGARAT motif, values within this range were obtained, leading to an average of 370 + 8° for these sites. Recently, a DNA bending angle of similar magnitude (400) was observed in the crystal structure of a X Cro -operator complex (Brennan et al., 1990). This value is in agreement with data obtained from a circular permutation assay using DNA fragments of similar length to those used in this study (Kim et al., 1989). This supports the validity of bend angles obtained by the measurement of position dependent mobilities.

From several experiments we calculated the apparent centre of bending to be located in the left half of both the canonical octamer (ATGCAAAT, Figure 4B) and the Ad2 octamer (ATGATAAT). This DNA region is mainly contacted by the POUs domain (Verrijzer et al., 1990b). Interestingly, in the TAATGARAT motif, the apparent bend centre is located in the GARAT half of this site (TAATGAGAT). This might well be related to the additional contacts observed for Oct-l in this region (Baumruker et al., 1988). To study the role of the two POU subdomains in bending, we purified the POUHD to near homogeneity from recombinant vaccinia virus infected HeLa cells (Verrijzer et al., 1990b) and assayed position dependent mobility. Because the binding affinity of the POUHD for the canonical octamer is too low, we employed the Ad2 octamer motif that has a relatively high binding affinity (KD = 2.7 x 10-9 M) for the POUHD. This enabled us to compare the effects of binding of the POU domain and the POUHD directly. In contrast to the POU -DNA complexes, the POUHD -DNA complexes showed no position dependent mobility, implying that the POUHD does not induce DNA bending (Figure 4A). From this result we infer that the POUs domain is responsible for DNA bending.

D

:..

.4,

.k

ALMN., ?-

:Ow '".., 11

0

.-K-i ft.

E

Ak ww

....

Fig. 4. DNA bending requires the POUS domain. (A) Gel electrophoresis of POU-DNA complexes with the ICP4 TAATGARAT motif (lanes I -8), the Ad2 octamer (lanes 9-16), the Ad4 octamer (lanes 17-24) and the POUHD complex with the Ad2 octamer (lanes 25-32). The circularly permutated fragments (A-H), described in Figure 1 were used. Due to the low affinity of the ICP4 site, a 4-fold higher amount of POU was added to obtain clear complex formation. Protein bound and free DNA (F) were separated on a 12% acrylamide gel. (B) Location of the apparent bend centre. The mobility of POU -DNA complexes is plotted against the position of the octamer within the fragment. The enzymes used to generate the restriction fragments from pOctaBend are indicated (A-H). A filled box shows the position of the octamer sequence '29ATTTGCAT136. The bend

centre is located around position 134.

3010

POU proteins bend DNA

The POU domain, but not the POUHD, enhances DNA cyclization Protein induced bending can also be monitored by the DNA cyclization assay (Kotlarz et al., 1986). If a protein bound to a linear DNA fragment bends the DNA, the end-to-end distance will decrease and hence, in the presence of ligase, the probability of ring closure will increase. In addition to the probability that the two ends are in close proximity, the cyclization kinetics of a linear fragment also strongly depend on the relative orientation of the two sticky ends (Shore and Baldwin, 1983). Thus, binding of a protein could influence the rate of cyclization by induction of a stable bend, or by changing the fractional twist i.e. the difference between the total helical twist and the nearest integer. We constructed CRIocta and CRIocta+4, two almost identical EcoRI fragments of 276 bp and 280 bp, respectively, in which the octamer site is located centrally.

A. 9..,

1-1

90 80

e

70

:

60

_

0 c. c

v.L

-

S~ ~ ~

50 4

401 2

I

I

The rate of cyclization was determined in the presence or absence of the Oct-I POU domain (Figure SA). In the absence of the POU domain a strong dependency on the fractional twist is found. The insertion of 4 bp in fragment CRIocta+4 leads to a 12-fold enhancement of the cyclization rate compared with CRIocta. When highly purified POU domain is added, the rate of cyclization of CRIocta is enhanced 1.7-fold and that of CRIocta+4 1.4-fold. Under the conditions used, - 50% of the DNA is bound by the POU domain, as determined by gel retardation. The intermolecular ligation reactions are not affected (not shown). Higher concentrations of the POU domain interfere both with the. intermolecular and the intramolecular ligation reaction, presumably by non-specific binding to DNA termini. Together with the binding kinetics of the POU domain, this provides only a narrow range of POU domain concentrations in which we could measure the effects on cyclization. Despite their different fractional twist, the POU domain enhances CRIocta and CRIocta+4 cyclization to an almost identical extent, indicating that local untwisting due to POU binding is negligible. The difference in POU stimulation between CRIocta and CRIocta+4 corresponds only to an untwisting of