Abstract
Histone deacetylase 2 (HDAC2) is one of the histone-modifying enzymes that regulate gene expression by remodeling chromatin structure. Along with HDAC1, HDAC2 is found in the Sin3 and NuRD multiprotein complexes, which are recruited to promoters by DNA-binding proteins. In this study, we show that the majority of HDAC2 in human breast cancer cells is not phosphorylated. However, the minor population of HDAC2, preferentially cross-linked to DNA by cisplatin, is mono-, di-, or tri-phosphorylated. Furthermore, HDAC2 phosphorylation is required for formation of Sin3 and NuRD complexes and recruitment to promoters by transcription factors including p53, Rb, YY1, NF-kappaB, Sp1, and Sp3. Unmodified HDAC2 requires linker DNA to associate with chromatin but is not cross-linked to DNA by formaldehyde. We provide evidence that unmodified HDAC2 is associated with the coding region of transcribed genes, whereas phosphorylated HDAC2 is primarily recruited to promoters.
Highlights
This post-translational modification enhances their enzymatic activity [7,8,9]
These results suggested that it was the phosphorylated form of Histone deacetylase 2 (HDAC2) that was principally associated with chromatin, posing the question as to the role and location of unmodified HDAC2 in chromatin
We have investigated the distribution of parent and phosphorylated HDAC2 forms in corepressor complexes and in chromatin
Summary
Cell Culture and Nuclear Extraction—Human breast cancer MCF-7 cells, HeLa cells and human embryonic kidney HEK293 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum as described previously [4, 9]. The proteins were boiled for 5 min in SDS loading buffer containing -mercaptoethanol at a final concentration of 5% (to reverse the DSP cross-linking), separated on SDS-10% polyacrylamide gels, transferred to nitrocellulose membranes, and immunochemically stained as indicated. The immunoprecipitate (IP) and equivalent volumes of lysate and immunodepleted (ID) fractions, corresponding to 0.02 A260 of lysate, were loaded onto SDS-10% polyacrylamide gels, transferred to nitrocellulose membranes, and immunochemically stained with anti-HDAC2, anti-HDAC1, or anti-CK2␣ antibodies. P53, YY1, and NF-B (p50, p65 subunits) are transcription factors known to achieve transcription silencing or dynamic acetylation/deacetylation by recruiting HDAC1 and 2 to specific promoters via Sin and/or NuRD complexes [23,24,25,26] To determine whether these transcription factors preferentially recruited phosphorylated HDAC2, we carried out immunoprecipitations of MCF-7 cell lysates with antibodies to these transcription factors.
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