H2A Case Study

Enhancer of Zeste Homolog 2 locus is intensely over expressed in breast and prostate cancer and it’s been established that its promoter inhibition by p53 has led to reduced cell proliferation and invasion (Bracken, 2003; Xiao, 2011). Objective is to clone a forward orientated EZH2 insert into a his-tagged pbluescript. Cloning EZH2 into a histidine-tagged pbluescript in a forward orientation potentially allows isolation of protein via Affinity Chromatography or Chromatin Immunoprecipitation therefore its role, effects and targets in the genome can be established. Resultant Recombinant plasmids in

The EZH2 (enhance of zeste homolog2) is an enzyme that in humans is fixed by EZH2 and its supply the information’s about making of enzyme called a histone me-thyltransferase. The ploycomb group (PcG) is a protein with the catalytic subunit of the PRC2 (polycomb repressive complex) which the transcriptional involved in maintaining the repressive of genes cells and also the EZH2 mainly performs as a silence gene. The EZH2 performs the role by addition of methyl groups on the ly-sine 27 of histone H3, a modification leading chromatin

Sufu has the ability to bind to GLI1, GLI2 and GLI3 proteins. It is theorized that GLI proteins are binded to Sufu in a head to tail orientation. GL1 is solely found in the nucleus, whereas Sufu can be found expressed in both the nucleus and the cytoplasm. However when they are both expressed together, Sufu brings the GLI1 into the cytoplasm which forces the nucleus to limit its transcription activity. The cytoplasm anchoring model suggests that GLI1 is anchored to the cytoplasm with the help of Sufu. Likewise, Sufu plays a similar role in Drosophilia by halting nuclear processes. Thus, it is found that Sufu travels back and forth between the cytoplasm and nucleus in both Drosophilia and mammals. A second model of Sufu function in mammals suggests the Sufu gene holds the ability of repressing transcription of GLIs. A study found that Sufu restricts GLI transcription by enlisting the help of the mSin3A HDAC corepressor complex. Sufu was also found to increase the ability of GLI1 to bind to DNA. Therefore, simply repressing the Sufu gene is enough to activate GLI transcription, alluding to its key role in the negative regulation of the SHH signaling

DNA is tightly coiled around core histone proteins in chromatin, forming nucleosomes. Linker histones bind nucleosomes together as chromatin. Acetylation, Methylation, phosphorylation, or ubiquitination of the N-terminal tails of each of the core histones affect the binding of the histone proteins to DNA. These modifications change the structure of the chromatin and allowing transcription factors accessibility to DNA regions. The combined effect of these modifications creates a histone code, which regulates gene expression. Of these various modifications, histone acetylation is the extensively studied, because of its role in the development and progression of tumors. Histone acetylation is controlled by histone acetyltransferases (HATs) and

During methylation, methyl groups (CH3) are attached to histone tails with the aid of histone methyltransferases (HMTs). These proteins transfer one, two, or three methyl groups from S-adenosyl-L-methionine to lysine or arginine residues. Lysine methylation involves a lysine methyltransferase that specifically targets and methylates lysine within the N-terminal tails. Similarly, arginine methylation occurs through the transfer of methyl groups from SAM to the ɯ-guanidino group of arginine. Although histone methylation does not change the charge of residues and does not greatly impact nucleosome interactions for chromatin folding, it results in binding sites for certain enzymes, which enables the regulation of chromatin condensation and nucleosome mobility. Methylation can also block the binding of proteins that interact with unmethylated histones and inhibit other modifications. Therefore, histone methylation regulates genes by promoting the binding of positive transcription factors and by blocking the attachment of negative transcription factors. However, some methylations can prevent gene transcription by mediating heterochromatin formation. For example, H3-K9 and H3-K27 methylation silences gene expression at certain euchromatic

Apart from PTMs of the H3 nucleosomes, the centromere code is also unique because CENP-A nucleosomes also undergo PTMs and regulate centromere function (Figure 1). The centromeric histone H4 of CENP-A containing nucleosomes undergo monomethylation at Lysine 20 following deposition of CENP-A into centromeres[94, 95]. When present in the H3 nucleosome, H4K20me1 is associated with transcribed regions throughout the genome[96]. Targeting the H4K20me1-specific demethylase PHF8 to centromeres reduces centromeric level of H4K20me1 and results in reduced recruitment of CENPT and CENP-H[94]. It is not yet clear whether this conserved H4K20me1 is directly responsible for the recruitment of the kinetochore components, or if it is an indirect effect caused by reduced

Investigation into the regulation of covalent histone modifications at enhancers and promoters has become a popular research interest due to the important role these modifications have on altering gene expression and modulating cell fate specification. Polycomb repressive complex 2 (PRC2) is a transcriptional repressive complex that consists of three components: enhance zeste 2 (EZH2), embryonic ectoderm development (EED) and suppressor of zeste 12 (SUZ12). The catalytic subunit, EZH2, can methylate lysine 27 of histone 3 (H3K27) to promote transcriptional silencing by facilitating chromatin compaction. Alterations of this complex have been

Since this is the only part of the histone protein which exists in an uncoiled state, it is subjected to far more modifications than the core proteins. Structurally, the H1 proteins exhibits a much longer C-terminal domain as compared to its N-terminal domain surrounding a highly conserved central globular domain. This C-terminal domain is arranged as a β hairpin motif associated with three α helices. The C-terminal is lined with Lysine residues with alternating Alanine residues. The amino acid constitution of the N and C terminals of this linker histone is detrimental for chromatin folding. The N-terminal region is composed of hydrophobic residues and the globular domain is made up of mostly basic amino acids; however the exact composition still remains to be characterized. Research has revealed the C-terminal domain by itself is able to bring about an efficient amount of chromatin fiber folding as the native H1 protein. Phosphorylation of this linker histone protein is the most studied post translational modification. Research has brought forward numerous examples of cross-talk found to be essential for regulation of this protein. Acetylation of Lysine at position 34 by HAT enzyme has been proven to be associated with activation of DNA

Using total internal reflection fluorescence (TIRF)-coupled photobleaching-assisted copy-number counting of single nucleosomes obtained from cultured cells, Padeganeh et. al. found that the majority of CENP-A nucleosomes contain CENP-A dimers[37, 42]. More recently, through the use of novel sequencing techniques, it was found that the CENP-A nucleosome protects a 133 bp genomic region consistent with octameric nucleosomes with DNA unwrapping at entry and exit[43]. While the existence of hemisomes (one molecule of each of CENP-A, H4, H2A and H2B forms nucleosomes) at specific cell cycle phases in flies, humans and yeast were proposed[44-47], predominant model of the CENP-A nucleosome is octameric structure. Such nucleosomes contain two molecules each of CENP-A, H4, H2A and H2B interspersed with canonical octameric H3 nucleosomes (Figure 1).

In Drosophila, nucleosomes form context-specific barriers to transcription that can be tuned at least in part by incorporation of H2A.Z (Weber et al., 2014). In murine embryonic stem cells, H2A.Z is enriched at active enhancers and promoters and facilitates chromatin accessibility to allow binding of a variety of active and repressive complexes required for self-renewal and differentiation (Hu et al., 2013). Also, transcription-coupled H2A.Z changes may play a role in cancer initiation and progression (Conerly et al., 2010). In human breast cancer cells, H2A.Z acts as an important player for enhancer functions. H2A.Z organizes a chromatin environment required for RNA polymerase II recruitment and enhancer-promoter(s) interactions, all essential features of enhancer activity (Brunelle et al., 2015). In Arabidopsis, H2A.Z predominantly associates with genes at euchromatic regions (Fig. 8A) (Coleman-Derr & Zilberman, 2012b; Sura et al., 2017; Zilberman et al., 2008). At highly expressed protein coding genes, H2A.Z is intensely enriched around the nucleosome-depleted region (NDR) at transcriptional start sites (TSSs), particularly at the +1 nucleosome (i.e. the first nucleosome downstream of the TSS) (Coleman-Derr & Zilberman, 2012b; Sura et al., 2017; Zhang et al., 2016; Zilberman et al., 2008) (Figure 8B). The +1 nucleosome can act as a barrier to

Previously, two nodes in the transcriptional network maintaining pluripotency were identified by Chen et al. 2008. Interestingly however, Zfp322a did not preferentially interact with either the c-Myc or Sox2/Oct4 transcription factor clusters. Zfp322a’s 10 C2H2 zinc finger domains bind the major groove of DNA (Lodish et al. 2013), and may give Zpf322a the ability to bind a diverse array of DNA sequences.  In support of this hypothesis, no DNA consensus sequence for Zfp322a binding was identified. Zfp322a co-localized equally with all 12 of the transcription factors tested. Coupled with Zfp322a’s dynamic ability to concomitantly repress differentiation, these observations suggest a more profound role for Zpf322a. From the genome-wide analysis of Zfp322a regulation the authors concluded that Zfp322a is an integral component in the pluripotency regulatory network.

ABSTRACT: An independently folding RNA G-quadruplex (GQ) forming domain interacts with the 40S ribosomal subunit and determines their binding affinity and function of an internal ribosomal entry site (IRES). The interaction begins to shed light to theprovide possible mechanistic role of RNA GQ structures in cap-independent translation initiation.

In a given cell order there are numerous types of sigma factors. Under normal condition or under stress different sigma factors recognize different promoter regions. Now that the holoenzyme is complete, the formation of the transcription bubble can take place. The RNA polymerase unwinds the DNA by itself. In order to start transcription energy from a ATP or GTP is required, where the ribose from the triphosphate provides a 3’ hydroxyl to attack the first phosphate in the first nucleotide, this displaces the pyrophosphate. The new RNA therefore has a triphosphate at its 5’ end. Once transcription has started the sigma factor falls off.

Cell volume is proportional to the amount of DNA in the cell nucleus. For example, doubling a cell's genome is expected to double the volume of space occupied by the chromosomes in the nucleus, but it causes only a 1.6-fold increase in the surface area of the nuclear envelope. This can disrupt the balance of factors that normally mediate interactions between the chromosomes and nuclear components, including envelope-bound proteins. The peripheral positioning of telomeric and centromeric heterochromatin may be disturbed as well, because there is less relative surface space on the nuclear envelope to accommodate this

Using a combination of IHC and IF experiments, we confirmed that the protein level of E2F3A and E2F8 during cell cycle, which reached peak at G1-early S and late S-G2, respectively, while E2F4 translocates from cytoplasm to nuclear when cells exit cell cycle. The activator E2Fs are required for transcriptional activation of S phase genes, while pocket protein needs to be phosphorylated by cyclin/CDK complexes to release its inhibitory effect on the activator E2Fs [10, 12, 29, 30]. The atypical repressor E2Fs function as transcriptional repressors of genes involved in DNA replication, inactivation of atypical repressor E2F activity would extend the progression of the DNA synthesis machinery [4, 5, 9, 16]. The typical repressor