Abstract
The constitutively nuclear histone deacetylases (HDACs) 1, 2, and 3 erase acetyl marks on acetyllysine residues, alter the landscape of histone modifications, and modulate chromatin structure and dynamics and thereby crucially regulate gene transcription in higher eukaryotes. Nuclear HDACs exist as at least six giant multiprotein complexes whose nonenzymatic subunits confer genome targeting specificity for these enzymes. The deacetylase activity of HDACs has been shown previously to be enhanced by inositol phosphates, which also bridge the catalytic domain in protein-protein interactions with SANT (Swi3, Ada2, N-Cor, and TFIIIB) domains in all HDAC complexes except those that contain the Sin3 transcriptional corepressors. Here, using purified recombinant proteins, coimmunoprecipitation and HDAC assays, and pulldown and NMR experiments, we show that HDAC1/2 deacetylase activity in one of the most ancient and evolutionarily conserved Sin3L/Rpd3L complexes is inducibly up-regulated by inositol phosphates but involves interactions with a zinc finger motif in the Sin3-associated protein 30 (SAP30) subunit that is structurally unrelated to SANT domains, indicating convergent evolution at the functional level. This implies that this mode of regulation has evolved independently multiple times and provides an evolutionary advantage. We also found that constitutive association with another core subunit, Rb-binding protein 4 chromatin-binding factor (RBBP4), further enhances deacetylase activity, implying both inducible and constitutive regulatory mechanisms within the same HDAC complex. Our results indicate that inositol phosphates stimulate HDAC activity and that the SAP30 zinc finger motif performs roles similar to that of the unrelated SANT domain in promoting the SAP30-HDAC1 interaction and enhancing HDAC activity.
Highlights
The constitutively nuclear histone deacetylases (HDACs) 1, 2, and 3 erase acetyl marks on acetyllysine residues, alter the landscape of histone modifications, and modulate chromatin structure and dynamics and thereby crucially regulate gene transcription in higher eukaryotes
Because the Sin3-associated protein 30 (SAP30) zinc finger (ZnF) motif has been shown previously to bind to molecules enriched in negatively charged moieties, we surmised that the SAP30 subunit might have a role akin to the SANT domains of MTA1 and SMRT corepressors in the NuRD and SMRT complexes (19 –21)
We asked whether fulllength HDAC1 or a construct lacking a large section of the unstructured C-terminal tail but retaining the catalytic domain, was sufficient for the interaction
Summary
Because the SAP30 ZnF motif has been shown previously to bind to molecules enriched in negatively charged moieties, we surmised that the SAP30 subunit might have a role akin to the SANT domains of MTA1 and SMRT corepressors in the NuRD and SMRT complexes (19 –21). To define the complementary protein surface involved in formation of the ternary complex, we mutated basic residues in HDAC1 that have been shown previously to bind to inositol phosphates, including Lys-31, Arg-270, and Arg-306, to glutamic acid both individually and in combination [21]. Our mutagenesis studies reveal that Arg-88, Ser, Ser-86, and Tyr-110 side chains in the SAP30 ZnF and His, Lys-31, Arg-270, and Arg-306 in HDAC1 are critical for the stability of the ternary complex In both proteins, the residues define contiguous surfaces for interactions with inositol phosphates and HDAC1 (Fig. 2D). The SAP30 ZnF R88E,R123E mutant produced little or no increase in these parameters relative to that measured for the enzyme alone These results indicate that the enhancement of HDAC1 deacetylase activity emanates from stable formation of the SAP30 ZnF–InsP6– HDAC1 ternary complex. EC50 parameters for HDAC1 activation by inositol phosphates Uncertainties correspond to standard deviations from three independent measurements
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