Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR.HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.     

Molecular characterization and association analysis of porcine interferon regulatory factor 1 gene

Interferon regulatory factor 1 (IRF1) is a member of IRF-family that was discovered to activate promoters in type I interferon (IFN) genes. It is shown to play functionally diverse role in the regulation of the immune system. In this report, the porcine IRF1 cDNA were cloned and a 7500 bp genomic DNA structure was identified. The putative IRF1 protein included 322 amino acids. Alignment and phylogenetic analysis of the predicted porcine IRF1 amino acids sequence with its homologies of other species show high identity (over 88%). Tissues expression of IRF1 mRNA was observed by RT-PCR, the results revealed IRF1 gene expressed widely in all analyzed tissues. Using the radiation hybrid panel, the porcine IRF1 gene was mapped to porcine chromosome 2 and closely linked to the locus IL4 (LOD = 7.09, 57cR). A SNP in exon2 of porcine IRF1 gene was demonstrated by sequencing and PCR–RFLP analysis. The further association analysis indicated that the SNP was significant associate with level of IFN-γ (day 20) in serum (P = 0.0001) and the ratio of IFN-γ to IL10 (day 20; day 35) in serum (P = 0.0165; P = 0.0095). The results suggested that the porcine IRF1 gene is strong candidate gene for these immune traits in pig.     

Cloning and promoter analysis of the chicken interferon regulatory factor-3 gene

Interferon regulatory factors (IRFs) are a family of DNA-binding proteins involved in mediating the cellular response to interferons (IFNs) and viral infection. Although extensively studied in mammals, IRFs of other vertebrates have been less well characterized. Previously, we cloned chicken interferon regulatory factor-3 (chIRF-3) mRNA, which is rapidly and transiently induced by double-stranded (ds)RNA. The chIRF-3 mRNA encodes a protein distinct from any known mammalian IRF. Here, we show that chIRF-3 is activated additively by type I and type II IFNs. To delineate the sequence elements required to regulate chIRF-3 expression, we cloned chlRF-3 and 0.48 kb of 5' flanking sequence. Computer analysis of the proximal promoter revealed three putative binding sites for nuclear factor (NF)-kappaB, two overlapping interferon-stimulated response elements (ISREs), and an interferon gamma activating sequence (GAS). The presence of both GAS and ISRE consensus sequences in the chIRF-3 promoter is unique among IRF family members. Both type I and II IFNs, as well as dsRNA and IRF-1, trans-activate the promoter in short-term transfection experiments. Mutational analysis of the promoter demonstrated that the putative NF-kappaB binding sites are needed for stimulation by dsRNA but not by either type I or type II IFN and that both the overlapping ISREs and GAS are required for full induction by type I or type II IFN.     

Activation of the human beta-interferon gene requires an interferon-inducible factor.

beta-Interferon (beta-IFN) gene expression can be induced by poly(I)-poly(C) or virus, but there is considerable variation in the extent of induction between different cell lines. We characterized two poorly inducible human cell lines, HeLa and 143 thymidine kinase negative (143 tk-), to define cellular factors involved in the activation of the beta-IFN gene. We show that the deficiency in beta-IFN induction in these cells can be complemented by fusion to highly inducible mouse cells. We conclude that the human cells are deficient in a trans-acting factor required for B-IFN gene activation. The level of induction of the beta-IFN gene in HeLa and 143 tk- cells can also be increased by priming with IFN before induction. If IFN priming is carried out in the presence of cycloheximide, a approximately 200-fold increase in induction is observed. We conclude that activation of the beta-IFN gene requires an IFN-inducible factor that is only expressed at low levels in unprimed HeLa and 143 tk- cells.     

Human interferon regulatory factor-1 gene and its promoter sequences revealed by population-based complete gene sequencing.

Interferon regulatory factor-1 (IRF-1) plays important roles in host immunity, cell proliferation and apoptosis. The current GenBank sequence for human IRF-1 (accession number: L05072) was derived from a human placenta DNA library and reported in 1992. In one recent population-based sequence study, we observed consistent discrepancies between our IRF-1 sequence data and GenBank reference sequences suggesting that, current IRF-1 reference sequence was not representative for all populations. By complete gene sequencing, we obtained a representative full-length IRF-1 sequence from a single subject. Compared to submission L05072, our population-based data contains: 35 nucleotide additions, 8 nucleotide removals and another 12 nucleotide replacements. A single nucleotide difference was observed in the IRF-1 promoter sequence compared to GenBank sequence (X53095). These changes were confirmed in 350 Kenyans and 28 non-African donors. The accuracy of a reference sequence is crucial for downstream genetic and functional studies and this study provides more complete and accurate data on the sequence of the human IRF-1 gene and its immediate promoter region.