A Positioned Nucleosome on the Human U6 Promoter Allows Recruitment of SNAPc by the Oct-1 POU Domain  Xinyang Zhao, P.Shannon Pendergrast, Nouria Hernandez  Molecular Cell  Volume 7, Issue 3, Pages 539-549 (March 2001) DOI: 10.1016/S1097-2765(01)00201-5

Figure 1 SNAPc and Oct-1 Are Localized on U6 and U1 Promoter Sequences In Vivo (A) Rapidly growing HeLa cells were treated with formaldehyde. Cross-linked chromatin was extracted, sonicated, and used as starting material for immunoprecipitations with beads coated with the antibodies indicated above the lanes or with just beads alone (lanes 13, 14, 21, 22). The immunoprecipitated material was analyzed by PCR with test (T) primers specific for the U6 promoter or control (C) primers hybridizing to a unique region 4 kb upstream of the U6 snRNA gene. (B) The experiment was as in (A) except that test (T) primers specific for the U1 promoter or control (C) primers hybridizing to a unique region 7 kb upstream of the U1 gene within the 45 kb U1 repeat (Bernstein et al., 1985) were used in the PCR. Different sets of test primers were used in lanes 1–16 and lanes 17–24 Molecular Cell 2001 7, 539-549DOI: (10.1016/S1097-2765(01)00201-5)

Figure 2 The DNA between the Octamer and the PSE in the U6 and U1 Promoters Is Resistant to MNase Digestion in Exponentially Growing Cells (A) Naked DNA (lane 1) or chromatin (lanes 2 and 3) was subjected to partial digestion with MNase. Double-strand cleavage sites were then mapped by ligation-mediated PCR with primers specific for the human U6 promoter. The main cleavage sites are indicated with arrowheads. The location of the PSE and octamer sequence relative to the labeled end of the primer (B) as well as the location of a putative nucleosome is indicated on the right. (B) Schematic representation of the U6 snRNA promoter. The hatched box on the left represents the unidirectional linker, and the small arrow represents the primer used in the second round of PCR. The label on the lower strand radioactive primer is marked with an asterisk. The distances separating the label end of the primer from the 5′ and 3′ edges of the octamer sequence and PSE are indicated. Major double-strand MNase cuts are indicated by arrowheads. (C) Naked DNA (lane 1) or chromatin (lane 2) was subjected to partial MNase digestion and analyzed as in (A) except that primers specific for the human U1 promoters were used. (D) Schematic representation of the U1 promoter. Symbols are the same as in (B) Molecular Cell 2001 7, 539-549DOI: (10.1016/S1097-2765(01)00201-5)

Figure 3 The Oct-1 POU Domain Binds to the Octamer Sequence in Chromatin Templates, but This Does Not Change the Nucleosome Pattern in the U6 Promoter Region (A) The U6 template pU6/Hae/RA.2 (lanes 1–4) or the same template containing a mutated octamer sequence (pU6/Hae/DSE−) (lanes 5–8) was assembled into chromatin (chrom) with S-190 extract from D. melanogaster postblastoderm embryos either without or with the Oct-1 POU domain derivatives indicated above the lanes. In lanes 9 and 10, naked (N) DNA template was used. The octamer sequence is indicated by a bracket. (B) pU6/Hae/RA.2 was assembled into chromatin with S-190 extract either without (lanes 1, 2, 5, and 6) or with (lanes 3, 4, 7, and 8) the Oct-1 POU domain. The chromatin was then subjected to partial MNase digestion for increasing amounts of time, fractionated on an agarose gel, transferred to a membrane, and visualized with a probe hybridizing to the U6 promoter (lanes 1–4) or to the β-lactamase gene in the plasmid (lanes 5–8). The lower panel shows the location of the probes, which are symbolized by short black bars and are separated by 1.7 kb Molecular Cell 2001 7, 539-549DOI: (10.1016/S1097-2765(01)00201-5)

Figure 4 The DNA between the Octamer and the PSE in pU6/Hae/RA.2 Assembled into Chromatin Is Resistant to MNase Digestion, Both with and without the Oct-1 POU Domain (A) Naked DNA (lane 5) or chromatin assembled without (lanes 1 and 2) or with (lanes 3 and 4) the Oct-1 POU domain was partially digested with MNase. Double-strand cleavage sites were then mapped by ligation-mediated PCR. The main cleavage sites are indicated with arrowheads. The location of the PSE and octamer sequence relative to the labeled end of the primer (B) as well as the location of a putative nucleosome are indicated on the right. (B) Schematic representation of the U6 snRNA promoter. Symbols are the same as in Figure 2B Molecular Cell 2001 7, 539-549DOI: (10.1016/S1097-2765(01)00201-5)

Figure 5 The Oct-1 POU Domain and SNAPc Bind Cooperatively to the Natural U6 Promoter When It Is Wrapped into Chromatin (A) A human U6 promoter probe was either assembled into a mononucleosome (lanes 1–4) or used as naked DNA (lanes 5–8) and incubated with the factors indicated above the lanes. The positions of free DNA, the mononucleosome (Nuc), the mononucleosome/Oct-1 POU domain complex (Nuc/POU), the mononucleosome/SNAPc complex (Nuc/SNAPc), the mononucleosome/Oct-1 POU domain/SNAPc complex (Nuc/POU/SNAPc), the naked DNA/SNAPc complex (SNAPc), and the naked DNA/Oct-1 POU domain/SNAPc (POU/SNAPc) complex are indicated. The bands labeled with a black dot correspond to naked DNA probe bound to one or more Oct-1 POU domains. These complexes were observed only when the naked DNA probe was limiting with respect to the Oct-1 POU domain. For lanes 5–8, a panel shows a low exposure of the bottom of the gel. (B) A mononucleosome probe as in (A) was either left untreated (lanes 1–4) or treated with the restriction enzyme Mse I (lanes 5–8). The probe was then incubated with the factors indicated above the lanes. The positions of the various complexes are indicated as in (A). (C) Schematic of the probe showing the location of Mse I cleavage sites Molecular Cell 2001 7, 539-549DOI: (10.1016/S1097-2765(01)00201-5)

Figure 6 Cooperative Binding on a Nucleosomal Template Requires the Same Protein–Protein Contacts as Cooperative Binding to Closely Spaced Octamer and PSE Sequences on Naked DNA A wild-type probe (lanes 1–16) or probes with a mutated PSE (lanes 17–20) or octamer sequence (lanes 21–24) were assembled into mononucleosomes and incubated with increasing amounts of the factors indicated above the lanes. E7R refers to the Oct-1 POU domain carrying the E7R mutation, and K900E refers to SNAPc carrying the K900E mutation in SNAP190. The complexes are labeled as in Figure 5A Molecular Cell 2001 7, 539-549DOI: (10.1016/S1097-2765(01)00201-5)

Figure 7 The Oct-1 POU Domain Activates U6 Transcription on a Chromatin Template (A) pU6/Hae/RA.2 (lanes 1–4) or the U6 template with a mutated octamer (construct pU6/Hae/DSE−, lanes 5–8) was mixed with S-190 extract without added histones and immediately incubated (as naked DNA template) in a HeLa cell extract depleted of endogenous Oct-1. The bands corresponding to U6 transcription (U6) or to the internal control (IC) are indicated. The numbers in the bracket below the lanes indicate amounts of U6 specific signal (normalized to the IC signal) relative to the signal obtained with the pU6/Hae/DSE− construct in the absence of the Oct-1 POU domain, which was set at 1 (lane 5). (B) pU6/Hae/RA.2 was assembled into chromatin with increasing amounts of the Oct-1 POU domains indicated above the lanes and then incubated with a HeLa cell extract depleted of endogenous Oct-1. The numbers in the bracket below the lanes indicate amounts of U6 specific signal (normalized to the IC signal) relative to the signal obtained in the absence of the Oct-1 POU domain, which was set at 1 (lane 1). (C) The pU6/Hae/DSE− (lanes 1–4) or pU6/Hae/RA.2 (lanes 5 and 6) constructs were assembled into chromatin with the Oct-1 POU domains indicated above the lanes and transcribed as in (B) Molecular Cell 2001 7, 539-549DOI: (10.1016/S1097-2765(01)00201-5)

Figure 8 A Positioned Nucleosome Mediates Cooperative Binding of the Oct-1 POU Domain and SNAPc on the Natural U6 Promoter The POUS and POUH subdomains of the Oct-1 POU domain are shown (details are discussed in the text) Molecular Cell 2001 7, 539-549DOI: (10.1016/S1097-2765(01)00201-5)