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Pei Zhang - Thesis Final Ver.pdf (23.55 MB)

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Identification of Replication-Dependent and Replication-Independent Linker Histone Complexes

Zhang, Pei, Zhang
http://rave.ohiolink.edu/etdc/view?acc_num=osu1468237233

Abstract Details

2016, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Histones are highly basic proteins that bind and package DNA into nucleosomes in the nucleus. Core histones (H2A, H2B, H3 and H4) form the octameric nucleosome core, whereas linker histones (H1 and H5) bind to the DNA entering and exiting the nucleosome core. There are 11 variants of linker histone H1 in mammalian cells: somatic replication-dependent variants (H1.1 to H1.5), somatic replication-independent variants (H1.0, H1x), and germ cell specific variants (H1t, H1T2m and HILS1 for testicular cells, and H1oo for oocytes) The sequences of these H1 variants vary greatly in their C-terminal domains. The individual functions of H1 variants are not fully understood. It was believed that H1 served as a global gene regulator by binding to chromatin non-specifically. However, although partially redundant in function, there is evidence indicating H1 variants have distinct roles in gene regulation and development. While deleting a single H1 variant might not lead to any observable phenotype, the H1.2/H1.3/H1.4 triple knockout mouse exhibited developmental defects and embryonic lethality. Microarray experiments showed that knockdown of each H1 variant altered a different subset of genes. The expression levels and activities of the H1 variants were also found to be highly regulated during cell differentiation and tumorigenesis. These observations suggest the hypothesis that each H1 variant has its individual function in the cells in addition to their roles as global chromatin modifiers. Studies on core histones indicate that replication-dependent and replication-independent histone variants can be involved in distinct protein complexes and assembly pathways. For example, histone H3.1 is assembled into chromatin in coordination with DNA replication during S phage, while another variant, H3.3, is exchanged throughout the cell cycle. The dynamics of H3.1 and H3.3 are mediated by distinct protein complexes that contain different chaperones for each of the H3 variants. All three subunits of histone chaperone CAF-1 are only found in the H3.1 complex, while another histone chaperone, HIRA, is only found in the H3.3 complex. CAF-1 and HIRA then dictate the replication-coupled and replication-independent assembly of these H3 variants. Currently it is not known whether the dynamics of replication dependent and independent variants of H1 are also regulated by distinct chaperones in a manner similar to H3.1 and H3.3 complexes. Previous studies proposed several linker histone chaperone candidates: Nucleosome Assembly Protein 1(NAP1), Nuclear Autoantigenic Sperm Protein (NASP), Nucleophosmin (NPM1), Prothymosin a (ProTa), and Template Activating Factor-I (TAF-1, also known as protein SET). The roles of these interactions between histone H1 and linker histone chaperones in H1 storage, chromatin assembly and disassembly are not fully understood. How the linker histone chaperones interact with each H1 variant also remains to be determined. To better understand the interations between H1 variants and other proteins, we studied their in vitro interactions with NASP, and found the H1 variants bind to NASP with different affinities. Fluorescence measurements of human H1 regulation of nucleosome dynamics and transcription factor binding within nucleosomes reveal that H1 variants function similarly to suppress transcription factor binding. We also purified protein complexes containing 6xHis-tagged H1 variants expressed in Tetracycline-inducible U2OS cell lines using column chromatography. Protein members of these complexes were identified by mass spectrometry analysis. We found replication-dependent and replication-independent H1 variants had distinct binding partners. For example, one binding partner of replication-dependent H1.1 and H1.2, nucleoprotein Tpr, was not found in the protein complex containing replication-independent H1x. Tpr knockdown leads to decreased levels of H1.1 and H1.2, but does not affect H1x levels. These findings suggest that H1 variants may perform non-redundant roles by associating with different proteins and forming distinct protein complexes.
Mark Parthun (Advisor)
Paul Herman (Committee Member)
Jeffrey Parvin (Committee Member)
Michael Freitas (Committee Member)
144 p.
Biochemistry
chromatin; histone; histone chaperone; linker histone; H1; Tpr

Recommended Citations

Citations

  • Zhang, Zhang, P. (2016). Identification of Replication-Dependent and Replication-Independent Linker Histone Complexes [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1468237233

    APA Style (7th edition)

  • Zhang, Zhang, Pei. Identification of Replication-Dependent and Replication-Independent Linker Histone Complexes. 2016. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1468237233.

    MLA Style (8th edition)

  • Zhang, Zhang, Pei. "Identification of Replication-Dependent and Replication-Independent Linker Histone Complexes." Doctoral dissertation, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1468237233

    Chicago Manual of Style (17th edition)

osu1468237233
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This open access ETD is published by The Ohio State University and OhioLINK.