Transcription through chromatin - link to diseases and therapeutics

The expression of chromosomal genes is regulated by posttranslational modification of both histone and nonhistone chromatin proteins and ATP-dependent remodeling of chromatin. Dysfunction of the modification and remodeling machineries can lead to several diseases, which include cancer, cardiac hypertrophy, and asthma. Many genetic diseases can also lead to malfunction of the machinery. The enzymes responsible for chromatin organization are the new targets for therapeutics. Inhibitors and activators of histone acetyltransferases and inhibitors of histone deacetylases may serve as new generation drugs

Analysis of cell proliferation rate in Oral Leukoplakia and Oral Squamous Cell Carcinoma

Objectives: Assessment of the cell proliferation rate in tissues can be one of the markers for impending malignancy in precancers. The state of activation and the proliferation activity of the cells can be assessed by the frequency of silver stained Nucleolar Organiser regions (AgNOR) within the nuclei which is significantly higher in malignant cells. The present study was carried out to analyze the distribution of the AgNOR in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC), and in their various histological grades, and to assess if the AgNOR distribution could give information on the malignant potentiality in premalignant lesions and aggressiveness of the malignant lesions. Study design: The study specimens comprised of 35 archival cases, of which 15 cases were of OL and 20 cases of OSCC. The specimens were stained by hematoxylin and eosin and modified silver staining method of Ploton et al. for the Nucleolar Organiser Regions. The specimens were analyzed independently by the two observers and was further statistically analysed. Results: The mean AgNOR count in OL was 2.80 ±0.50 and in cases of OSCC was 5.71± 1.08. The mean AgNOR count in OL cases of mild dysplasia was 2.59 ±0.66, in moderate dysplasia was 2.92± 0.43 and in severe dysplasia was 2.79. The mean AgNOR count in cases of well differentiated OSCC was 5.73± 1.62 and in cases of moderately differentiated OSCC was 5.67±1.19. Conclusion: The mean AgNOR count was higher in cases of OSCC as compared to cases of OL, and the AgNOR counts increased with the increase in the grades of dysplasia indicating a higher proliferative rate with increase in dysplasi

The use of hyperbaric oxygen therapy in actinomycotic osteomyelitis associated florid cemento osseous dysplasia: report of a familial case.

Florid Cemento-Osseous Dysplasia (FCOD) is a well recognized fibro-osseous disease of the jaws commonly seen in the middle aged African women, although it may occur in the Caucasians and Asians. There is a reported incidence of less than 2% in the Indian population. The clinical and the radiographic features of FCOD may overlap with features of chronic diffuse scle-rosing osteomyelitis, which makes diagnosis of the disease difficult in symptomatic cases. Moreover the jaws may be susceptible to osteomyelitis, which may mask the underlying FCOD in some cases. The case reported here is that of a 56 year old Indian female patient with long standing FCOD invol-ving the maxilla and the mandible, who was asymptomatic till all her teeth were extracted for fabrica-tion of complete dentures. The patient experienced symptoms of pain, swelling and purulent discharge soon after the use of dentures and was treated by surgical removal of the affected bone. The same symptoms recurred in other areas of the jaw over a period of time and these areas were treated surgica-lly. The patient subsequently presented with pain in the mandibular left posterior region of ten days duration, and was diagnosed as FCOD with superimposed Actinomycotic Osteomyelitis based on his-topathology and Gram’s stain, and was treated by surgical removal of the affected bone followed by hyperbaric oxygen therapy (HBO). The radiograph of the surgical site showed good amount of bone regeneration within six months of HBO therapy without recurrence of the FCOD in the same area till date

Epigenetic Modulation of miR-122 Facilitates Human Embryonic Stem Cell Self-Renewal and Hepatocellular Carcinoma Proliferation

The self-renewal capacity ascribed to hESCs is paralleled in cancer cell proliferation, suggesting that a common network of genes may facilitate the promotion of these traits. However, the molecular mechanisms that are involved in regulating the silencing of these genes as stem cells differentiate into quiescent cellular lineages remain poorly understood. Here, we show that a differentiated cell specific miR-122 exemplifies this regulatory attribute by suppressing the translation of a gene, Pkm2, which is commonly enriched in hESCs and liver cancer cells (HCCs), and facilitates self-renewal and proliferation. Through a series of gene expression analysis, we show that miR-122 expression is highly elevated in quiescent human primary hepatocytes (hPHs) but lost or attenuated in hESCs and HCCs, while an opposing expression pattern is observed for Pkm2. Depleting hESCs and HCCs of Pkm2, or overexpressing miR-122, leads to a common deficiency in self-renewal and proliferation. Likewise, during the differentiation process of hESCs into hepatocytes, a reciprocal expression pattern is observed between miR-122 and Pkm2. An examination of the genomic region upstream of miR-122 uncovered hyper-methylation in hESCs and HCCs, while the same region is de-methylated and occupied by a transcription initiating protein, RNA polymerase II (RNAPII), in hPHs. These findings indicate that one possible mechanism by which hESC self-renewal is modulated in quiescent hepatic derivatives of hESCs is through the regulatory activity of a differentiated cell-specific miR-122, and that a failure to properly turn “on” this miRNA is observed in uncontrollably proliferating HCCs

Genome-Wide Analysis of KAP1 Binding Suggests Autoregulation of KRAB-ZNFs

We performed a genome-scale chromatin immunoprecipitation (ChIP)-chip comparison of two modifications (trimethylation of lysine 9 [H3me3K9] and trimethylation of lysine 27 [H3me3K27]) of histone H3 in Ntera2 testicular carcinoma cells and in three different anatomical sources of primary human fibroblasts. We found that in each of the cell types the two modifications were differentially enriched at the promoters of the two largest classes of transcription factors. Specifically, zinc finger (ZNF) genes were bound by H3me3K9 and homeobox genes were bound by H3me3K27. We have previously shown that the Polycomb repressive complex 2 is responsible for mediating trimethylation of lysine 27 of histone H3 in human cancer cells. In contrast, there is little overlap between H3me3K9 targets and components of the Polycomb repressive complex 2, suggesting that a different histone methyltransferase is responsible for the H3me3K9 modification. Previous studies have shown that SETDB1 can trimethylate H3 on lysine 9, using in vitro or artificial tethering assays. SETDB1 is thought to be recruited to chromatin by complexes containing the KAP1 corepressor. To determine if a KAP1-containing complex mediates trimethylation of the identified H3me3K9 targets, we performed ChIP-chip assays and identified KAP1 target genes using human 5-kb promoter arrays. We found that a large number of genes of ZNF transcription factors were bound by both KAP1 and H3me3K9 in normal and cancer cells. To expand our studies of KAP1, we next performed a complete genomic analysis of KAP1 binding using a 38-array tiling set, identifying ~7,000 KAP1 binding sites. The identified KAP1 targets were highly enriched for C2H2 ZNFs, especially those containing Krüppel-associated box (KRAB) domains. Interestingly, although most KAP1 binding sites were within core promoter regions, the binding sites near ZNF genes were greatly enriched within transcribed regions of the target genes. Because KAP1 is recruited to the DNA via interaction with KRAB-ZNF proteins, we suggest that expression of KRAB-ZNF genes may be controlled via an auto-regulatory mechanism involving KAP1

Sole-Search: an integrated analysis program for peak detection and functional annotation using ChIP-seq data

Next-generation sequencing is revolutionizing the identification of transcription factor binding sites throughout the human genome. However, the bioinformatics analysis of large datasets collected using chromatin immunoprecipitation and high-throughput sequencing is often a roadblock that impedes researchers in their attempts to gain biological insights from their experiments. We have developed integrated peak-calling and analysis software (Sole-Search) which is available through a user-friendly interface and (i) converts raw data into a format for visualization on a genome browser, (ii) outputs ranked peak locations using a statistically based method that overcomes the significant problem of false positives, (iii) identifies the gene nearest to each peak, (iv) classifies the location of each peak relative to gene structure, (v) provides information such as the number of binding sites per chromosome and per gene and (vi) allows the user to determine overlap between two different experiments. In addition, the program performs an analysis of amplified and deleted regions of the input genome. This software is web-based and automated, allowing easy and immediate access to all investigators. We demonstrate the utility of our software by collecting, analyzing and comparing ChIP-seq data for six different human transcription factors/cell line combinations

Characterization of the Contradictory Chromatin Signatures at the 3′ Exons of Zinc Finger Genes

The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3′ exons of zinc finger genes (ZNFs) are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither bioinformatic nor experimental approaches support the hypothesis that the 3′ exons of ZNFs are promoters. We further characterized the histone modifications at the 3′ ZNF exons and found that these regions also contain H3K36me3, a mark of transcriptional elongation. A genome-wide analysis of ChIP-seq data revealed that ZNFs constitute the majority of genes that have high levels of both H3K9me3 and H3K36me3. These results suggested the possibility that the ZNF genes may be imprinted, with one allele transcribed and one allele repressed. To test the hypothesis that the contradictory modifications are due to imprinting, we used a SNP analysis of RNA-seq data to demonstrate that both alleles of certain ZNF genes having H3K9me3 and H3K36me3 are transcribed. We next analyzed isolated ZNF 3′ exons using stably integrated episomes. We found that although the H3K36me3 mark was lost when the 3′ ZNF exon was removed from its natural genomic location, the isolated ZNF 3′ exons retained the H3K9me3 mark. Thus, the H3K9me3 mark at ZNF 3′ exons does not impede transcription and it is regulated independently of the H3K36me3 mark. Finally, we demonstrate a strong relationship between the number of tandemly repeated domains in the 3′ exons and the H3K9me3 mark. We suggest that the H3K9me3 at ZNF 3′ exons may function to protect the genome from inappropriate recombination rather than to regulate transcription

Functional analysis of KAP1 genomic recruitment and investigation of its transcriptional regulatory effects on the genome

In mammalian cells, multiple cellular processes such as gene silencing, cell growth, cell differentiation, maintenance of pluripotency, neoplastic transformation, DNA repair and apoptosis converge on the evolutionarily conserved protein KAP1 (KRAB-associated protein1). Despite the advances made through biochemical and other in vitro studies on KAP1, not much was known about (1) its in vivo binding sites, (2) its mechanism of recruitment to the genome or (3) its effect on the transcriptional output of its target genes. Presented within is a summary of my dissertation research which attempts to answer these three questions about KAP1 and ultimately to understand how KAP1 mediates diverse effects on cell biology. As a first step towards identifying the in vivo genomic binding sites of KAP1, I performed ChIP-chip experiments in Ntera2D1 cells using an antibody targeted against KAP1 in combination with tiling arrays that spanned the entire human genome, leading to the identification of ∼7000 KAP1 binding sites. Subsequently, using a combination of ChIP-chip and ChIP-seq techniques, I identified KAP1 targets in numerous other cell types, including both normal and tumor cells. I hypothesized that there are two mechanisms of KAP1 recruitment to DNA. To test this hypothesis, I created a series of FLAG-tagged KAP1 mutants and stably introduced them into HEK293 cells. Each mutant inactivated a different functional domain of the KAP1 protein; specifically I mutated the N-terminal RBCC domain (required for interaction with KRAB-ZNFs), the C-terminal PB domain (required for interaction with chromatin modifying enzymes), and the HP1 binding domain (required for interaction with HP1). I performed ChIP-seq experiments using an antibody that recognizes the FLAG tag to test the requirement of each domain of KAP1 for genomic recruitment. My experiments (i) prove that interaction of KAP1 with KRAB-ZNFs is required for a subset of targets, (ii) suggest the presence of a hitherto uncharacterized mechanism of KAP1 recruitment to its promoter bound targets, and (iii) reveal a previously unknown role for HP1 in tethering KAP1 to DNA. I performed ChIP-seq experiments using an antibody that recognizes the FLAG tag to test the possible role of each ZNF in recruiting KAP1 to its genomic binding sites. Surprisingly, although the KRAB-ZNFs interacted with endogenous KAP1, the ChIP-seq assay was not able to detect any genomic binding sites for these proteins. It is possible that the chosen KRAB-ZNF proteins have a role in mediating interaction between KAP1 and RNA or other non-chromatin proteins, and therefore their function cannot be elucidated using the ChIP-seq assay. It is also possible that the selected KRAB-ZNF proteins bind DNA only upon certain developmental or environmental triggers, and hence ChIP-seq could not identify any genomic targets for these proteins under the given conditions. To address the question of how KAP1 was recruited to its promoter targets, I used a bioinformatics approach to identify highly enriched transcription factor binding motifs within the central 100 bp of the top 100 promoters bound by KAP1. This approach identified the CCGGAA motif; this motif is generally used by the ETS family of transcription factors, and ELK4 specifically binds to a very similar sequence as found in the KAP1 promoter peaks. Therefore I hypothesized that ELK4 may be involved in the recruitment of KAP1 to promoters. I then experimentally tested this prediction by performing ChIP-seq for ELK4 in HEK293 cells and showed that out of 1084 promoter-bound targets of KAP1, 358 (∼33%) co-localized with ELK4 bound sites, suggesting that ELK4 may play an important role in recruiting KAP1 to this category of genomic binding sites. Finally, to address the role of KAP1 as a global regulator of transcription, I assessed the mRNA levels of its target genes in cells deficient for KAP1 using Illumina gene expression arrays and RNA-seq. Surprisingly, I showed that there is no correlation between the enrichment of KAP1 at its strongest binding sites (ZNF 3' ends) and the expression of the bound ZNF genes. Also there was no change in mRNA splicing or processing of KAP1-bound ZNF genes, which are enriched for the heterochromatic mark H3me3K9 even in KAP1-deficient cells. (Abstract shortened by UMI.

Hemifacial Microsomia - A Case Report

Hemifacial Microsomia is a disorder characterised by the anomalies of the face, ears, eyes and the vertebrae. The incidence of this disorder is 1:3000 to 26, 000 and is usually seen at birth. It may be familial or sporadic cases. The disorder is more common in males especially on the right side of the face. The disorder was first identified by M. Goldenhar and J. Genet Hum in 1952, and was called Oculo-Auriculo-Vertebral dysplasia by R. J. Gorlin. Clinically one sees facial asymmetry, macrostomia, micrognathia, and microtia to anotia of the external ear, preauricular skin tags, and Epibulbar tumors in the eyes. One such case of Hemifacial Microsomia seen in a boyaged 9 years, who complained of facial asymmetry on the right side of the face since birth and an extra auricle on the same side, is reported here