Using the Origin Recognition Complex to Identify Histone Modifications in Mammalian Cells
The DNA has developed the capacity to stimulate replication at numerous positions on every chromosome in multicellular organisms. These several replication origins differ in terms of size, structure, temporal regulation, and firing efficiency. Most of these dissimilarities are believed to be affected by epigenetic factors, such as phosphorylation, methylation, acetylation, sumoylation, and ubiquitination. For instance, histone deacetylases have been found to have intense impacts on replication timing and initiation frequency. Mechanism of identifying the origin of DNA replication have been discovered. However, how proteins interact to promote this replication initiation is still unclear. Thus, this experiment seeks to determine if ORC can be used to detect histone modifications in DNA replication origins. We hypothesize that the ORCA-ORC complex can be used to recognize histone modifications in heterochromatin and euchromatin DNA of mammalian cells in the late S phase and early S phase, respectively.
Identifying the origin recognition complex (ORC) in the baker’s yeast opened up a path that led to various investigations on DNA replication in multicellular organisms. In the last two decades, researchers have been comprehensively studying the regulation of replication and amplification in a wide range of organisms, illuminating that DNA replication is instigated by a systematic assemblage of the pre-replication complex (pre-RC). The origins of DNA replication consist of conserved A, B1, and B2 elements (Chen et al., p.1). The role of element A and part of element B1 is to outline the binding sequence for the ORC, which is considered to bind in an ATP-dependent manner (Kawakami et al., p.1). Additionally, ORC engages in recruiting other vital proteins, including Cdc6, Cdt1, and the presumptive DNA helicase MCM, to the autonomously replicating sequence (ARS), forming a pre-replicative complex (pre-RC) before the DNA replication begins (Chen et al., p.1). ORC contains six proteins: Orc1, Orc2, Orc3, Orc4, Orc5, and Orc6 (Li, and Stillman, pg. 3). Orc1 and Orc5 bind to ATP, while the biggest five subunits are projected to have an AAA fold and a DNA-binding winged-helix domain (WHD) in their C-terminal halves (Chen et al., p.1).
Chromatin environment influences the replication timing as well as origin firing frequency; for instance, replication start earlier in S phase for bulk euchromatin compared to heterochromatin (Zhou et al. pg. 2). Histone deacetylases have been found to have intense impacts on replication timing and initiation frequency (Zhou et al. pg. 2). For example, loss of yeast rpd3 histone deacetylase was observed to speed the firing time of replicating origins in the late S-phase. On the other hand, loss of the NAD-dependent histone deacetylase Sir2 was observed to suppress a mutation in CDC6, indicating the blocking of pre-replication development (Zhou et al. pg. 2). The deletion of Sir2 resulted in an increased number of origins. Furthermore, inhibition of histone deacetylases in eukaryotic cells was reported to change the origin utilization. Consequently, histone acetylation enables the pre-replication complex development and early S phase commencement at numerous replication origins of multicellular organisms. Various studies regarding the replication initiator proteins have revealed the mechanism of recognizing replication origins; however, the manner in which different initiator proteins interact to support the initiation of DNA replication is still unclear (Chen et al., p.1). The complex structure, as well as the chromatin packaging of the chromosomes of eukaryotes, have been considered an issue concerning fast and precise DNA replication. As a result, the DNA has developed the capacity to instigate replication at numerous positions on every chromosome. These numerous replication origins differ in size, structure, temporal regulation, and firing efficiency. Many of these variations are believed to be influenced by epigenetic factors, such as phosphorylation, methylation, acetylation, sumoylation, and ubiquitination (Zhou et al. pg. 1). In this experiment, we hypothesize that the ORCA-ORC complex can be used to recognize histone marks in heterochromatin and euchromatin DNA of mammalian cells in the late S phase and early S phase respectively. Therefore, the experiment aims to test whether the origin recognition complex can recognize histone modifications associated with initiation sites of DNA replication.
Aim 1: To determine if ORCA co-localizes with DNA replication origins in mammalian cells to confirm ORCA is associated with initiation sites of DNA replication in mammalian cells.
Aim 2: To test if there is an interaction between the ORCA and ORC at the origin sites of DNA replication in mammalian cells during replication.
Aim 3: To determine the presence and loci of the histone marks such as H3K9me3, H3K27me3, and H3K9ac, in the heterochromatin and euchromatin DNA in the late S phase and early S phase of mammalian cells replication.
To determine whether ORCA co-localizes with DNA replication origins, the replication sites will be mapped through nascent strand sequencing. This method is highly used to identify origins of DNA replication and involves isolating the total DNA, denaturing it, and subjecting it to fractionation (Kunnev et al., pg. 1; Smith et al., pg. 3). In multicellular organisms, the commencement of DNA replication necessitates the assemblage of a multiprotein pre-replicative complex (pre-RC) (Popova et al. pg. 2). ORC-associated protein (ORCA/ LRWD1) interacts with the ORC and is associated with heterochromatin formation to facilitate pre-RC assembly in mammalian cells, (Shen et al., pg. 1). Therefore, ORCA is expected to co-localize with the DNA replication origins.
Regarding the second aim, a biochemical analysis will be carried to recognize proteins associated with ORC and contributing to the DNA origin specification. This will be determined through immunoprecipitation (IP), and the IP materials will be subjected to mass spectrometric analysis. Immunoprecipitation with ORCA pAb in human cells demonstrates the association with ORCA with ORC subunits (Shen et al., pg. 3). ORCA interacts with the ORC complex and is considered to stabilize ORC on the chromatin and loss of ORCA in primary fibroblasts has been associated with being arrested in G1 in addition to having reduced MCM loading, indicating ORCA is vital un replication origin licensing (Wang et al. pg.5). ORCA also controls the heterochromatin organization in a manner that is independent of its regulation in replication initiation.
The analysis of histone modifications on specific genome loci will be determined through chromatin immunoprecipitation (ChIP) using an antibody directed against the site-specific modifications (Rizzardi et al. pg.2). Chromatin will be prepared from formaldehyde-fixed cells because core histones are firmly attached to DNA. Micrococcal nuclease treatment or sonication can be used to fragment the cells and then histone-DNA complexes can be immunoprecipitated using the specific antibody. Then, co-immunoprecipitated DNA is subjected to deep sequencing (seq), microarray analysis, or quantitative PCR (qPCR) (Kimura, pg.5). ORCA interacts with ORC, Cdt1, and geminin with maximum level during G1 and progressive reduction as the cells enter the S phase (Shen et al. pg. 1). ORCA-ORC exists in a single complex with histone methyltransferases, which are considered to play a role in heterochromatin-specific histone modifications (Suter et al., pg. 2). ORCA is believed to interact with repressive histone marks such as H3K9me3 and H3K27me3 (Kimura, pg.4). Thus, H3K9me3, H3K27me3, among other histone marks that are associated with repressive genes in heterochromatin are expected to be found in the late S phase (Kim, and Kim, pg. 1), while active genes, such as H3K27ac and H3K4me3, are expected to be observed in euchromatin DNA in the early S phase (Tang et al., pg. 2).
One problem that might arise from the experiment is two overlapping modifications on the same loci. This is a problem that can arise from issues in handling chromatic from cell populations. Another limitation of this study would be the quality of the antibody used. The antibody must be specific and applicable to ChIP. Antibodies used must to differentiate small modifications, such as single methylation on the same lysine (Kimura, pg.5). Specificity can be increased by using monoclonal antibodies.