Cloning and characterization of the histone-fold proteins YBL1 and YCL1

Abstract

Histones are among the most conserved proteins in evolution, sharing a histone fold motif. A number of additional histonic proteins exist and are involved in the process of transcriptional regulation. We describe here the identification, cloning and characterization of two small members of the H2A-H2B sub-family (YBL1 and YCL1) related to the NF-YB and NF-YC subunits of the CCAAT-binding activator NF-Y and to the TATA-binding protein (TBP) binding repressor NC2. Unlike the latters, YBL1 and YCL1 have no intrinsic CCAAT or TATA-binding capacity. In nucleosome reconstitution assays, they can form complexes with histones in solution and on DNA and they are part of relatively large complexes, as determined by glycerol gradient experiments. Our data support the idea that YBL1 and YCL1 are divergent with respect to NF-YB and NF-YC for specific functions, but have coevolved the capacity to interact with nucleosomal structures.

Figure 1

Sequence comparisons of YBL1 and YCL1. (A) Alignments of YBL1 and YCL1 sequences from mouse (GenBank accession no. AA839465 for YBL1, AI195704 and AI931369 for YCL1), man (AW246427 for YBL1, R55966 for YCL1), rat (AA818074 for YBL1), zebrafish (AI416300 for YBL1, AI437035 for YCL1), chicken (AI980453 for YCL1), Drosophila (AI237921 for YBL1, AI061700 for YCL1), B.mori (AU003161 for YBL1) and D.discoideum (C83909 for YBL1). (B) Alignments of H2B and H2A-like sequences. §, α-Helical regions; *, loops; S (blue), s (green) and P (red), surface, self and Pair residues, respectively, as discussed by Arents and Moudrianakis (3); I, identity; :, similarity.

Figure 2

Expression of YBL1. (A) Northern blot analysis using 10 µg of total RNA or 3 µg of polyA+ RNA with the indicated cell lines. (B) Western blot analysis using 10 µg of nuclear extracts from the indicated cell line. An anti-NF-YB antibody was used to normalize protein concentrations.

Figure 3

Heterodimerization of YBL1–YCL1. (A) Recombinant His-tagged YBL1 was renatured with YCL1 and purified over nickel NTA-agarose. L, load; FT, flow through; W1, 0.3 M KCl wash; W2, 1 M KCl wash; E, eluates with 0.3 M imidazole. Lower panel, YCL1 was processed without His–YBL1. (B) Same as (A), except that His–YCL1 was used with nuclear extracts from CH27 as a source of YBL1, NF-YB and NF-YC. The proteins were revealed by western blots with the indicated antibodies.

Figure 4

YBL1–YCL1 is not a CCAAT or TATA interacting dimer. (A) EMSA analysis of decreasing concentrations (300, 100, 30, 10, 3, 1 and 0.3 ng) of NF-YB–NF-YC (lanes 1–7) or YBL1–YCL1 (lanes 8–14), in the presence of 100 ng of NF-YA and a labeled CCAAT-box oligonucleotide (MHC class II Ea Y box). (B) Immunoprecipitations with an anti-NF-YA monoclonal-Mab7 or a control (anti-GATA) antibody after incubation of NF-YA with YBL1–YCL1 (upper panel) or NF-YB–NF-YC (lower panel). L, load; U, unbound; E, bound. (C) Same as (A), except that the oligonucleotide used was AdML TATA box with decreasing concentrations of YBL1–YCL1 (100, 30, 10 and 3 ng) and TBP (100 ng) in lanes 2–5, or without TBP (lanes 6–9). Control incubations with purified endogenous TFIIA (lanes 10 and 11) or recombinant TFIIB (lanes 12 and 13) were run with (lanes 10 and 12) or without TBP (lanes 11 and 13).

Figure 5

Interactions of YBL1–YCL1 with histones. (A) Coomassie blue stained SDS gel of recombinant histones purified from inclusion bodies. (B) His–YCL1 was incubated with the indicated histones, renatured and passed over a nickel NTA-agarose column. Gels were stained with Coomassie blue. (C) Same as (B), except that His–YBL1 was used.

Figure 6

Nucleosome reconstitutions with YBL1–YCL1. (A) Increasing concentrations of YBL1–YCL1 dimer (0.3, 1 and 3 µg) were used with 1 µg of the indicated H2A–H2B, H3–H4 combinations. Arrows, complexes formed with histones; *, faint complexes generated by YBL1–YCL1. (B) Antibody challenge of EMSA complexes containing YBL1–YCL1. H3–H4 tetramers (lanes 1–6) and nucleosomes (lanes 7–12) were reconstituted without YBL1–YCL1 (lanes 1–3 and 7–9) or with (lanes 4–6 and 10–12) in the absence of antibodies (lanes 1, 4, 7 and 10) or with anti-YBL1 (lanes 2, 5, 8 and 11) or anti-GATA1 control antibodies (lanes 3, 6, 9 and 12). In lanes 13–15, YBL1–YCL1 only was used in reconstitutions and similarly challenged with antibodies. (C) MNase I digestions of H3–H4 tetramers (lane 2), H3–H4–YBL1–YCL1 (lane 3), nucleosome (lane 4) and nucleosome–YBL1–YCL1 (lane 5). F, free DNA digested with Mnase I; arrows, increased sensitivity sites generated by the presence of YBL1-YCL1; *, protections.

Figure 7

YBL1–YCL1 is found in relatively large complexes. Fractions from glycerol gradients with CH27 nuclear extracts were checked in a western blot with the anti-YBL1 antibody (upper panel). The molecular weight markers, run on parallel gradients, are indicated. Similarly, fractions generated from a gradient containing only the recombinant YBL1–YCL1 dimer were checked in westerns and shown in the lower panel. In the first lane 10 ng of recombinant YBL1 was loaded.