Identification of Novel Interacting Proteins of Histone Gene Regulator, HINF-P: a Dissertation
Graduate School of Biomedical Sciences
Repressor Proteins; Cell Cycle Proteins; Gene Expression Regulation; Histones; Nuclear Proteins; S Phase; Academic Dissertations
Histone Nuclear Factor P (HiNF-P) is a known transcriptional regulator that is critical for the activation of replication dependent histone H4 genes during S phase. HiNF-P is a 65 kDa zinc finger protein that binds to its consensus binding sequence in the Cell Cycle Control Element (Site II) of the proximal promoter region of 11 of the 14 histone H4 genes. HiNF-P is a known co-factor of the global histone gene regulator and cyclinE/CDK2 substrate p220NPAT, however it was not known if this regulatory function reflected a physical interaction. In addition, other HiNF-P interacting proteins have yet to be identified. The work presented in this thesis identifies and characterizes HiNF-P interactions with various proteins within the cell, including p220NPAT. A yeast two-hybrid interaction screen identified candidate interacting proteins of HiNF-P and provided insight into novel cellular functions and transcriptional targets. A candidate yeast two-hybrid approach identified an interaction between HiNF-P and p220NPAT. This direct physical interaction links the cyclin E/CDK2 signaling pathway governing the G1/S phase transition with replication dependent histone gene transcription in S phase.
An unbiased yeast two-hybrid screen for HiNF-P interacting proteins revealed an interactome library which suggests roles of HiNF-P in multiple cellular processes. This screen identified 67 candidate HiNF-P interacting proteins that are RNA processing factors, known and putative gene regulators, uncharacterized proteins, proliferation related proteins, as well as metabolic and signaling proteins. Identification of multiple RNA binding and processing factors, including the splicing cofactor, SRm300, links HiNF-P to mRNA processing. HiNF-P is potentially functioning in mRNA processing by interacting with these proteins directly and functioning in complex with them, or more likely, by recruiting these and other splicing factors to sites of transcription.
We identified a number of known and putative gene regulators which are candidate HiNF-P interacting proteins. We isolated the atypical C2CH zinc finger protein, THAP7, a known transcriptional repressor. THAP7 interacts with HiNF-P by co-immunoprecipitation and co-immunofluorescence experiments. We show forced expression of THAP7 abrogates HiNF-P/p220 mediated activation of histone H4 gene transcription. THAP7 may represent a novel co-factor of HiNF-P and p220 mediated regulation of histone H4 genes. Identification of interacting proteins of HiNF-P that are involved in transcriptional regulation provides insight into other transcriptional targets of HiNF-P. HiNF-P is localized throughout the nucleus, presumably at multiple gene foci. These interacting proteins may represent novel co-factors of HiNF-P regulation of these other multiple target genes.
HiNF-P has been identified as a regulator of cell cycle dependent histone genes, therefore we were interested in identifying other proliferation related proteins with which HiNF-P is interacting. We identified a number of proteins thought to be involved in cellular proliferation, including Ki-67 and an unknown protein XTP2. The functions of these proteins have not been identified. An interaction with HiNF-P might suggest a role for these proteins in histone gene regulation. In addition, Ki-67 has been implicated transcriptional control of ribosomal genes, although no role of HiNF-P in this function has been identified.
HiNF-P is a known regulator of histone gene expression via a functional interaction with the global histone gene regulator and cyclin E/CDK2 substrate, p220. This thesis demonstrates HiNF-P directly interacts with the N-terminus of p220. This interaction requires multiple regions within the N-terminus including a LisH-like domain known to function in protein-protein interactions, a region (aa 121-145) known to be required for histone gene transactivation, and another uncharacterized region (209-318). In addition a phylogenically conserved region within the C-terminus of HiNF-P, the HiNF-P Specific Conserved Region (PSCR) is necessary for this interaction. Mutational analysis of these regions abrogates this interaction. HiNF-P and p220 co-localize at specific foci within the cell corresponding to Cajal bodies, which are known sites of histone gene clusters. This work shows that this interaction is necessary for histone gene transcriptional activation and HiNF-P dependent recruitment of p220 to histone H4 gene promoters. In addition HiNF-P as well as p220 interact with the Stem Loop Binding Protein (SLBP) and co-localize in situ. SLBP is a necessary factor for histone pre-mRNA processing events which also occur at Cajal bodies. These interactions provide evidence of the coupling of transcription and processing of histone genes and the involvement of common factors in both processes. This would allow for rapid production of abundant histone proteins which is needed during S phase. This thesis has identified multiple candidate interacting proteins of HiNF-P. These proteins establish HiNF-P as a protein involved in many cellular processes and mechanisms beyond transcriptional control of cell cycle dependent histone genes.
Miele, A. Identification of Novel Interacting Proteins of Histone Gene Regulator, HINF-P: a Dissertation. (2006). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 300. https://escholarship.umassmed.edu/gsbs_diss/300
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