Incorporation Of Histone H3 Research Articles

Abstract 458: Characterization of ATRX polyclonal, application of immunohistochemical analysis on astrocytoma and glioblastoma

Abstract Introduction: Alpha thalassemia/mental retardation syndrome X-linked (ATRX) plays a role in chromatin regulation and maintenance of telomeres. It regulates incorporation of histone H3.3 into telomeric chromatin. ATRX is also a major component of various essential cellular pathways such as DNA replication and repair, chromatin higher-order structure regulation, and gene transcriptional regulation. ATRX loss was observed in grades II/III astrocytomas, oligoastrocytomas, oligodendrogliomas, and glioblastomas. In grades II/III gliomas, most ATRX-loss cases had IDH1/2 mutations. Assessment of ATRX loss by IHC staining captures the majority of mutations, giving a reasonable sensitivity in neuropathology diagnostics. In this study, the specificity and sensitivity of ATRX were analyzed on multiple tissues focusing on Astrocytoma and Glioblastoma tissues. Materials and Methods: IHC staining has been performed by using formalin-fixed paraffin-embedded tissue with standard deparaffinization steps and a heat-induced retrieval method using a citrate buffer solution in a pressure cooker (Decloaking chamber). Antibody was incubated for 30 minutes, followed by HRP detection and DAB solution for visualization. Slides were stained using semi-automated staining system (intelliPATH). ATRX polyclonal antibody [Biocare Medical, California, USA] diluted in a carrier at an optimal concentration with standard protocol. Results: ATRX stained 44% of diffuse and anaplastic astrocytoma and 57% of glioblastoma. Also, cross-reactivity testing determined ATRX polyclonal may stain normal brain in some cases. A more comprehensive data evaluation shown below: Conclusions: ATRX polyclonal shows a potential advantage when diagnosing brain tumors (especially astrocytoma and glioblastomas) even if some cross reactivity can be observed. ATRX in conjunction with other brain marker antibodies can offer a better tool in diagnosing brain tumors. Diseased Tissue Specificity and SensitivityTissuePositive CasesTotal Cases% +Astrocytoma163644Glioblastoma4757Breast Cancer132748Colon Cancer84020Lung Cancer245246Prostate Cancer164238Normal Tissue Cross-ReactivityCerebrum51145Cerebellum2367 Citation Format: Jose Jonathan Diaz, Tatiana Scoggin, Jason Ramos. Characterization of ATRX polyclonal, application of immunohistochemical analysis on astrocytoma and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 458.

Investigating the Effects of Histone H3.3 Point Mutations in Neural Crest Cells and Craniofacial Development

Craniofacial development is an intricate process that requires the meticulous and coordinated expression of many genes. Histone proteins which are fundamental to both the structural integrity and organization of DNA into nucleosomes and consequently, chromatin play an important role in coordinated gene expression. Furthermore, the modification of histone tails is now known to be an essential mechanism for coordinated expression of a large number of genes. Recently, a point mutation in the gene h3f3a which encodes the histone variant Histone H3.3, was identified in a forward genetic screen in zebrafish. H3.3A was found to be important for neural crest cell induction and generation of cranial neural crest cell derived head bones. The authors proposed that incorporation of histone H3.3 was required for expression of genes important for cranial neural crest cell specification, whereas remodelling of this histone tail may be important for later stages in neural crest cell differentiation. However, it remains to be confirmed if disrupted modification of histone tails is also important in neural crest cells. We postulate that modification of the H3.3 tail is required for coordinated expression of genes important for neural crest migration and/or differentiation. To test this hypothesis, we used CRISPR/Cas9 to generate a mouse line with a conditional missense mutation in H3f3a. The mutation generated was previously shown to result in an abnormal increase in methylation of K27 and a decrease in methylation of K36 of the histone H3.3 tail. In this poster, we will present preliminary data using the Wnt‐1 Cre2 mouse line to drive expression of this dominant negative mutation in neural crest cells. Alcian Blue Staining will be used to analyze cartilage in E14.5 embryos and alizarin red will be used to stain bones formed in heads of E17.5 embryos. Bones will be analyzed in control and mutant embryos. Our result will enable us to uncover if K27 or K36 methylation is important in neural crest cell formation, migration and/or differentiation. This work will begin to unravel the role of epigenetics in neural crest cell formation.Support or Funding Information

Incorporation of histone H3.1 suppresses the lineage potential of skeletal muscle.

Lineage potential is triggered by lineage-specific transcription factors in association with changes in the chromatin structure. Histone H3.3 variant is thought to play an important role in the regulation of lineage-specific genes. To elucidate the function of H3.3 in myogenic differentiation, we forced the expression of GFP-H3.1 to alter the balance between H3.1 and H3.3 in mouse C2C12 cells that could be differentiated into myotubes. GFP-H3.1 replaced H3.3 in the regulatory regions of skeletal muscle (SKM) genes and induced a decrease of H3K4 trimethylation (H3K4me3) and increase of H3K27 trimethylation (H3K27me3). Similar results were obtained by H3.3 knockdown. In contrast, MyoD-dependent H3.3 incorporation into SKM genes in fibroblasts induced an increase of H3K4me3 and H3K27me3. In mouse embryos, a bivalent modification of H3K4me3 and H3K27me3 was formed on H3.3-incorporated SKM genes before embryonic skeletal muscle differentiation. These results suggest that lineage potential is established through a selective incorporation of specific H3 variants that governs the balance of histone modifications.

HIRA dependent H3.3 deposition is required for transcriptional reprogramming following nuclear transfer to Xenopus oocytes

BackgroundNuclear reprogramming is potentially important as a route to cell replacement and drug discovery, but little is known about its mechanism. Nuclear transfer to eggs and oocytes attempts to identify the mechanism of this direct route towards reprogramming by natural components. Here we analyze how the reprogramming of nuclei transplanted to Xenopus oocytes exploits the incorporation of the histone variant H3.3.ResultsAfter nuclear transplantation, oocyte-derived H3.3 but not H3.2, is deposited on several regions of the genome including rDNA, major satellite repeats, and the regulatory regions of Oct4. This major H3.3 deposition occurs in absence of DNA replication, and is HIRA-and transcription-dependent. It is necessary for the shift from a somatic- to an oocyte-type of transcription after nuclear transfer.ConclusionsThis study demonstrates that the incorporation of histone H3.3 is an early and necessary step in the direct reprogramming of somatic cell nuclei by oocyte. It suggests that the incorporation of histone H3.3 is necessary during global changes in transcription that accompany changes in cell fate.