Regulation of gene expression is required for embryogenesis and maintenance of the highly specialized and diverse neuron populations of the retina. Chromatin remodelling proteins control gene expression by modifying chromatin structure and are essential for many biological processes including mammalian development. The ATP-dependent chromatin remodelling protein Snf2h is highly expressed in the central nervous system, and pathogenic variants that cause neurodevelopmental abnormalities in the human population have recently been identified. This work aims to characterize the effects of Snf2h loss in the retina. Snf2h retinal conditional knockout (cKO) mice were generated using Snf2h-floxed mice and Chx10-Cre retina-specific Cre driver lines to ablate the Snf2h protein from the retina at embryonic day 10.5. Visual function was assessed via optomotor response-based testing and full-field scotopic electroretinography, and histological changes were examined via immunohistochemistry. Disease progression was tracked at one, two, three, and six months of age. Snf2h cKO mice showed a significant decline in visual function and exhibited retinal neuron loss compared to wildtype control littermates at all time points assessed. This work shows that the chromatin remodelling protein Snf2h plays an essential role in the structure and function of the retina.

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The purpose of the present thesis was to examine and quantitatively study key postnatal events in the developing and mature human cerebral cortex within the context of the systematic variation in the laminar structure of the cortex that underlies connectivity patterns and cortical function. To address this, we expanded upon previous work completed by LeRoy Conel and performed extensive analysis of Cresyl-Violet, Cajal, and Golgi-Cox images of various Von-Economo’s Areas for five age groups in order to see where variations in dendritic tree arborization, which is visualized by Golgi-Cox staining, pruning, which is visualized by Cresyl Violet staining, and cytoskeletal changes, which are visualized by Cajal staining, may lie. The results of this analysis showed that across all age groups and cortical types, there are consistently high levels of dendritic tree arborization, cytoskeletal changes, and cell body density changes in all layers across younger age groups, followed by slight declines in older age groups, following the trend of rapid gray matter expansion during the first two years of postnatal life before shifting towards white matter growth. These levels were also the most exaggerated in cortical layers III and V of Agranular, Dysgranular, Eulaminate II, and Eulaminate III areas, suggesting that cortical layers III and V of these areas change the most rapidly during the key events of postnatal development that Conel sought to analyze in his staining experiments. We also found that in layers III and V, Golgi-Cox values increased, reflecting the growth of dendritic trees while Cresyl-Violet and Cajal values decreased, reflecting decreases in cell body density, allowing for increased space between cells, highlighting a complementary inverse relationship that is seen between neurite outgrowth and cell body density. Since postnatal developmental processes in the cortex are largely driven by transcription factors that regulate chromatin expression well into adulthood, we also examined how certain epigenetic modifications in the nuclei of neurons in the adult cortex may explain for variations seen between areas in the analysis of Conel’s images. To address this, we analyzed populations of H4K12 and SC35 expressing pyramidal neurons in layers III and V of Area 46 and Area 32 in order to further understand how patterns of acetylation and RNA splicing may play a role in the rapid changes of layers III and V seen in Eulaminate II and Dysgranular areas. In the case of SC35, higher levels of splicing were seen in layer III of Area 32 and layer V of Area 46, suggesting that splicing centers within the neurons of these layers and areas are more organized. In the case of H4K12, similar levels of acetylation were observed in layers III and V of Area 46 whereas higher levels were observed in layer V of Area 32, indicating a certain degree of regulation of gene expression within these areas. The results of this study demonstrate variations in the timing of the major events of postnatal development across layers in different structural types of cortical areas that correspond to limbic, multimodal, and highly specialized unimodal areas and the inverse relationship seen between neurite outgrowth and changes in cell body density. Additionally, these results demonstrate the impact that patterns of RNA splicing and acetylation may have on these events and can be utilized to identify disruptions in developmental disorders such as autism. / 2024-09-21T00:00:00Z

<p>Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition.</p><p>We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing.</p><p>By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.</p>

Background: In all higher organisms, the nuclear DNA is condensed into nucleosomes that consist of DNA wrapped around a core of highly conserved histone proteins. DNA bound to histones and other structural proteins form the chromatin. Generally, only few regions of DNA are accessible and most of the time RNA polymerase and other DNA binding proteins have to overcome this compaction to initiate transcription. Several proteins are involved in making the chromatin more compact or open. Such chromatin-modifying proteins make distinct post-translational modifications of histones – especially in the histone tails – to alter their affinity to DNA. Aim: The main aim of my thesis work is to study the targeting of chromatin modifiers important for correct gene expression in Drosophila melanogaster (fruit flies). Primary DNA sequences, chromatin associated proteins, transcription, and non-coding RNAs are all likely to be involved in targeting mechanisms. This thesis work involves the development of new computational methods for identification of DNA motifs and protein factors involved in the targeting of chromatin modifiers. Targeting and functional analysis of two chromatin modifiers, namely male-specific lethal (MSL) complex and CREB-binding protein (CBP) are specifically studied. The MSL complex is a protein complex that mediates dosage compensation in flies. CBP protein is known as a transcriptional co-regulator in metazoans and it has histone acetyl transferase activity and CBP has been used to predict novel enhancers. Results: My studies of the binding sites of MSL complex shows that promoters and coding sequences of MSL-bound genes on the X-chromosome of Drosophila melanogaster can influence the spreading of the complex along the X-chromosome. Analysis of MSL binding sites when two non-coding roX RNAs are mutated shows that MSL-complex recruitment to high-affinity sites on the Xchromosome is independent of roX, and the role of roX RNAs is to prevent binding to repeats in autosomal sites. Functional analysis of MSL-bound genes using their dosage compensation status shows that the function of the MSL complex is to enhance the expression of short housekeeping genes, but MSL-independent mechanisms exist to achieve complete dosage compensation. Studies of the binding sites of the CBP protein show that, in early embryos, Dorsal in cooperation with GAGA factor (GAF) and factors like Medea and Dichaete target CBP to its binding sites. In the S2 cell line, GAF is identified as the targeting factor of CBP at promoters and enhancers, and GAF and CBP together are found to induce high levels of polymerase II pausing at promoters. In another study using integrated data analysis, CBP binding sites could be classified into polycomb protein binding sites, repressed enhancers, insulator protein-bound regions, active promoters, and active enhancers, and this suggested different potential roles for CBP. A new approach was also developed to eliminate technical bias in skewed experiments. Our study shows that in the case of skewed datasets it is always better to identify non-altered variables and to normalize the data using only such variables.

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