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Chromatin is made of DNA and a variety of proteins and this is the material that makes up chromosomes. There is many ways that this chromatin can be organized. The organization of chromatin is one factor that makes the function of a chromosome so
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  Jason Simonides MCB 3841W 9/27/17 Chromatin is made of DNA and a variety of proteins and this is the material that makes up chromosomes. There is many ways that this chromatin can be organized. The organization of chromatin is one factor that makes the function of a chromosome so complex. Centromere  protein A (CENP-A) is a variant of histone H3 and is very important when looking at centromere targeting. CENP-A chromatin in humans resides in repetitive DNA alpha satellite sequences. In the  Homo sapiens  chromosome 17 (HSA17), this region of alpha satellite DNA where centromeric chromatin is localized is called D17Z1. Pericentromeric chromatin is characteristic of heterochromatin and therefore is tightly packed. Pericentromeric chromatin surrounds CENP-A chromatin, limiting centromeric nucleosomes to this D17Z1 region. When this pericentromeric chromatin is not present and the alpha satellite sequences are directly next to loosely packed euchromatin, CENP-A can spread into the short arm (p arm) of the chromosome. The study of HSA17 showed the plasticity of the human centromeric chromatin through fluorescence and sequencing techniques. CENP-A is normally localized at D17Z1 but is capable of spreading when a deletion occurs on the HSA17 removing most of the centromeric region and half of the p arm. This breakpoint is thought to be caused by long interspersed nuclear elements (LINEs). This  plasticity of the human centromeric chromatin raised questions about whether the spreading of CENP-A to atypical locations had an effect on the genome. One effect of CENP-A spreading included repression of the ELAC2 gene, a gene located in euchromatin that controls transcription. Another effect on the genome included invasion of CENP-A chromatin into regions of open and transcribed chromatin, such as DNase hypersensitivity sites (DSS), which  can lead to cancer. In addition, CENP-A spreading could lead to neocentromeres and dicentric chromosomes that can give rise to developmental defects and cancer. The general structure of chromosome HSA17 from fibroblast line GM08148 was examined using fluorescence in situ hybridization (FISH) with DNA probe p17H and DAPI. The normal HSA17 was stained to show where the centromeric region lies on the chromosome. This centromeric region included D17Z1 flanked by its pericentromeric regions D17Z1-B and D17Z1-C. Looking at HSA17, centromeres are targeted to D17Z1. In a study of the naturally occurring human chromosome 17 mutant, a portion of HSA17 was deleted and was a supernumerary marker (mar17). This deleted portion included most of the D17Z1 region, the  pericentromeric region and part of the p arm of the chromosome. The remaining chromosome was the HSA17 with the deletion and labeled del(17). The specific probe that was used was specific to D17Z1 alpha satellite array on HSA17. FISH showed the centromeric regions on these corresponding pieces by targeting D17Z1 alpha satellite array sequences. On HSA17 the alpha satellite fluorescence localized to the D17Z1 region. On mar17 the fluorescence showed the deleted region was mostly centromeric as almost all of mar17 was glowing bright red with fluorescence. There was not much fluorescence displayed in del(17), indicating that most of the centromeric sequence was deleted from the HSA17. The breakpoint at which the deletion occurred was identified as a 292 kb region using BAC CGH arrays. PCR with primers to STS markers was then used to narrow down this  breakpoint region to between D17S2117 and D17S1808, which was a 28 kb region. The absence of bands on the gel was indicative of a deletion at that specific location. The presence of bands on the gel showed that there was still D17Z1 region at the particular spot and therefore could not  be identified as the deleted region. There was a faint band at the D17S2117 region due to some  of the D17Z1 region being in tact on del(17) after the deletion. At the specific region where the  break occurred there were L1/LINEs observed. Due to these LINEs being present, these retroelements are thought to have contributed to the breakpoint at this region. These LINEs repeated throughout this region and are suspected to have created an area of instability. At this  breakpoint, del(17) was left lacking several features that the HSA17 would have. More specifically, 12.9 Mb of HSA17, 2.5 Mb of D17Z1 alpha satellite DNA, around 500 kb of  pericentromeric chromatin, and 9.9 Mb of the short arm of the chromosome directly next to the  break. All of these features are found in mar17 observed in the FISH but not found in del(17). Del(17) was mapped using fiber immunostaining and FISH. This compared the overlap of CENP-A with the region D17Z1. In a normal HSA17 chromosome, the CENP-A overlapped completely with the D17Z1 region. The pericentromeric region was in tact in the HSA17 region and therefore the CENP-A was contained within this region. Del(17), lacking pericentromeric chromatin, showed partial overlap of CENP-A with the D17Z1 region but 45% of this chromatin was not localized here. This indicated that it might be localized in nearby non-satellite DNA. This non-satellite DNA includes euchromatin and would be uncharacteristic for CENP-A to localize here and could result in genomic instability. The specific location of this new centromeric location was on the p arm of del(17). It is important to note that once a centromere is established at a particular point on a chromosome, that centromere will be maintained at that location. Therefore, there was evidence of a neocentromere when CENP-A spread because there is now a new centromeric region on a chromosome at a location that is not normally centromeric. CENP-A spread into the p arm of del(17) and was observed through chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR). Based on the results, due to the deleted portion of the chromosome, it confirmed the data from the immunostaining  and FISH mapping, which showed CENP-A spreading. Six genomic sites on the chromosome were tested for the percent of D17Z1 enrichment because D17Z1 is normally where CENP-A would localize. The higher the percentage of D17Z1 there was, the more enrichment of CENP-A. Sites 1-5 were distal to the centromeric region and located on the short arm of del(17). Site 6 was a control that should have showed no enrichment of D17Z1 because this specific genomic site was on the deleted region and not included in del(17). At these five specific genomic sites, the enrichment of D17Z1 was compared between HSA17 and del(17). There was less enrichment of D17Z1 at the specific genomic sites on HSA17 than the enrichment of D17Z1 on del(17).  Normally, there would be little to no sequences of D17Z1 at sites 1-5 on HSA17 because these are located distally on the chromosome and are representative of euchromatin on the p arm of the chromosome. Due to this fact, it was significant that the enrichment of D17Z1 was higher in del(17) compared to HSA17 looking at sites 1-5. Furthermore, the D17Z1 genomic site on del(17) showed less enrichment of D17Z1 than the enrichment of D17Z1 on HSA17. It was concluded that CENP-A spreading occurred due to the deletion. On del(17), the CENP-A spread out over the p arm of the chromosome showing less enrichment in the actual centromeric region, represented by the enrichment of D17Z1 at the D17Z1 genomic site and consequently more enrichment distally in the euchromatin region. The euchromatin region where CENP-A was enriched shared a region with DSS. These DSS are indicative of open chromatin where CENP-A can invade. Due to this spreading of CENP-A into these regions, there is genomic instability that can lead to cancer. This is because the neocentromere is now maintained at a location where there is active gene regulation occurring. To show the effect of CENP-A spreading into the euchromatin region of the chromosome, the relative expression of ELAC2 was compared  between HSA17 and del(17). There was less relative expression of ELAC2 on del(17) by more  than 50% compared to a normal HSA17 chromosome. CENP-A chromatin spread into the euchromatin and reduced expression of this ELAC2 gene that would normally be important in aiding in transcription. In this study, the ELAC2 gene was tested but it is believed that more neighboring genes could also be affected by CENP-A spreading. If this is the case, then it is  possible that developmental defects and cancer could ensue. The absence of pericentromeric chromatin due to the deleted region of the HSA17 chromosome caused for CENP-A spreading. The breakpoint of the specific deletion was likely caused by LINEs, which made the chromosome genetically unstable at this point. At this  breakpoint there was a loss of pericentromeric chromatin. Therefore, CENP-A spread from the normal alpha satellite DNA region known as D17Z1 into the p arm of del(17), which was characterized by euchromatin. The higher enrichment of CENP-A in the euchromatin regions made these regions unstable because there was less expression of normal functioning genes at these sites, such as the ELAC2 gene. To go from here, research will look at the chromatin structure surrounding the new location of the centromeric chromatin as well as further investigate the importance of the pericentromeric heterochromatin regions around the centromeric region. The research will give new insights to centromeric function as well as what happens when it is repositioned in a chromosome.
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