Dissertation/Thesis Abstract

Recruitment of HCFC1 to Chromatin by THAP11 and ZNF143 Transcription Factors
by Vinckevicius, Aurimas, Ph.D., Northwestern University, 2016, 152; 10117187
Abstract (Summary)

Control of cell cycle progression is an important aspect of normal cell biology and is frequently disrupted in cancers. HCFC1 has been established as a critical component of cell cycle regulation, but the mechanism by which it mediates this function is not clearly understood. Recently HCFC1 has been shown to directly interact with the E2F family of proteins, which are key regulators of cell cycle progression, and bind promoters of cell cycle control genes. Interestingly, some of these promoters are also bound by THAP11, another HCFC1-interacting protein, which may be responsible for HCFC1 recruitment to these loci.

In this study, we analyzed existing genome-wide data and performed bench experiments at a large number of HCFC1 target genes to determine the mode of HCFC1 recruitment to cell cycle control genes. These analyses revealed that, on a genome-wide scale, HCFC1 is highly associated with THAP11 and ZNF143 transcription factors on chromatin, while the association with E2F1 – a member of the E2F protein family – is significantly weaker. Furthermore, the results of our study indicate that HCFC1 recruitment to chromatin is conditional on THAP11 and ZNF143, but not E2F1. Contrary to current literature, which suggests that THAP11 and ZNF143 may compete for binding to chromatin, our observations indicate that THAP11, ZNF143, and HCFC1 chromatin occupancy is mutually dependent.

Genome-wide and in vitro studies demonstrate that THAP11 and ZNF143 recognize overlapping DNA sequences. Given our results above, the mechanism of cooperative binding by these two proteins is unclear and warrants further investigation. To avoid possible artifacts associated with in vitro binding experiments, we used chromosomally-integrated synthetic constructs and CRISPR-Cas9-mediated approaches in intact cells to elucidate the role of DNA sequence in recruitment of the THAP11/ZNF143/HCFC1 complex and to establish its biological relevance. We show that the ACTACA submotif, shared by both THAP11 and ZNF143, directs recruitment of THAP11 and HCFC1 to ZNF143-occupied loci. Importantly, its position, spacing, and orientation relative to the ZNF143 core motif are critical for this action. Biologically, CRISPR-Cas9-mediated alteration of the ACTACA submotif in the endogenous OPHN1 promoter abolished its transcriptional activity and lead to lower RNA Polymerase II and histone H3 lysine 4 tri-methyl levels. Our in vivo approaches provide strong evidence for the molecular role of the ACTACA submotif in THAP11, ZNF143, and HCFC1 cooperative recruitment to chromatin and its biological necessity for target gene expression.

Indexing (document details)
Advisor: Chakravarti, Debabrata
Commitee:
School: Northwestern University
Department: Life Sciences
School Location: United States -- Illinois
Source: DAI-B 77/10(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology
Keywords: Crispr, E2F, HCFC1, THAP11, ZNF143
Publication Number: 10117187
ISBN: 978-1-339-78590-5
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