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

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

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

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

Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells

We compared 12 different cell populations, including embryonic stem cells before and during differentiation into embryoid bodies as well as various types of normal and tumor cells to determine if pluripotent versus differentiated cell types use different mechanisms to establish their transcriptome. We first identified genes that were not expressed in the 12 different cell populations and then determined which of them were regulated by histone methylation, DNA methylation, at the step of productive elongation, or by the inability to establish a preinitiation complex. For these experiments, we performed chromatin immunoprecipitation using antibodies to H3me3K27, H3me3K9, 5-methyl-cytosine, and POLR2A. We found that (1) the percentage of low expressed genes bound by POLR2A, H3me3K27, H3me3K9, or 5-methyl-cytosine is similar in all 12 cell types, regardless of differentiation or neoplastic state; (2) a gene is generally repressed by only one mechanism; and (3) distinct classes of genes are repressed by certain mechanisms. We further characterized two transitioning cell populations, 3T3 cells progressing from G0/G1 into S phase and mES cells differentiating into embryoid bodies. We found that the transient regulation through the cell cycle was achieved predominantly by changes in the recruitment of the general transcriptional machinery or by post-POLR2A recruitment mechanisms. In contrast, changes in chromatin silencing were critical for the permanent changes in gene expression in cells undergoing differentiation