Identification of CECR2-containing chromatin-remodeling complexes and their Chromatin-binding sites in mice

• Author / Creator

  Hassanzadeh Niri, Farshad

• Eukaryotic nuclear DNA is packaged into chromatin, a complex nucleoprotein structure. This has functional consequences by controlling the accessibility of DNA to binding factors responsible for many important cellular processes such as transcription, DNA replication and DNA repair. ATP-dependent chromatin remodeling complexes such as the ISWI family can regulate these cellular processes by altering the chromatin structure. CECR2 is a chromatin remodeling factor that forms a complex with ISWI proteins SNF2H and SNF2L. Loss-of-function mutations in Cecr2 result in the perinatal lethal neural tube defect, exencephaly. Nonpenetrant animals that survive to adulthood exhibit subfertility. CECR2 loss affects transcription of multiple genes and is also involved in γ-H2AX formation and DSB repair. The mutant phenotypes indicate that CECR2 plays an important role in neural tube development and reproduction, but the mechanism of its function is not known. I therefore have investigated the components of the CECR2 complex and its chromatin binding sites in ES cells and testis. I hypothesized that the CECR2 complexes contain tissue-specific components that may correspond to tissue-specific functions in ES cells and testis. I also hypothesized that the CECR2 containing complexes occupy different chromatin binding sites. This work first required the development of a highly specific CECR2-specific antibody. I confirmed that CECR2 forms a complex with SNF2H and SNF2L both in mouse ES cells and in testes. I showed that the CECR2-containing complex in mouse ES cells and testes has a size of approximately 2 MDa, suggesting the presence of additional components in the complex. Mass spectrometric analysis of CECR2-containing complexes revealed novel binding partners of CECR2 in ES cells and adult testis. I identified CCAR2 as a new member of the CECR2-containing complex in ES cells and possibly testis. CCAR2 has been shown to be involved in DNA damage response, which is also a known function of CECR2. Strikingly, there is a difference in the composition of complexes isolated from ES cells and adult testis. LUZP1 (leucine zipper protein 1) was confirmed to be a binding partner of CECR2 only in mouse ES cells and not in testes. Luzp1 mutant mice display exencephaly in 42% of embryos, indicating its role in neural tube closure. LUZP1 appears to play a role in stabilizing the CECR2 complex. I showed that the CECR2 complexes have different components, which opens the door toward understanding the multiple functions of CECR2. Disruption of Cecr2 results in dysregulation of expression of many genes in mouse embryos, however the direct transcriptional targets of the CECR2 complexes are unknown. Therefore, to find the direct binding sites of the CECR2 complex, chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis was performed in ES cells and adult testis. Looking at the overlapping binding sites of CECR2 and SNF2H allowed for a more powerful analysis and investigating the additional overlap with the binding sites of LUZP1 in ES cells allowed me to look for the ES cell-specific binding sites. Little overlap of CECR2 binding sites between ES cells and testes was observed, suggesting tissue-specific transcriptional regulation. Analysis of the ChIP-seq data revealed that the CECR2 complex occupies the promoter and cis-regulatory regions of many genes. The identified genes in ES cells are involved in different aspects of embryonic development including brain, heart and kidney development. The Cecr2 mutants exhibit abnormalities in these three organs. Gene ontology (GO) analyses of the genes associated with the binding sites of the CECR2 complex in ES cells showed that this complex modulates important molecular pathways including Shh and Wnt signaling. The genes identified in testes are involved in different aspects of reproduction and development. I identified many candidate genes that can be used to investigate the CECR2 function in neurulation and fertility. Hsd17b2, Lpar1, Nf, Lrp6 and Phactr4 are possibly directly regulated by the CECR2 complex, all of which cause exencephaly when mutated. Elmo1, Fgfr4, Ggt1, Insr, Itgb3 and Schip1 from the ES cell dataset and Cdc14b, Nfia, Pcsk1 and Styx from the testis dataset are candidate genes involved in reproduction. My findings revealed that there are ES and testis-specific CECR2 complexes in mice. I showed that these complexes have different compositions and chromatin binding sites, which will facilitate understanding the multiple functions of CECR2 during development and reproduction.

• Subjects / Keywords

  • CECR2

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3CZ32M99

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.