Transcriptional Dynamics During Lens Fiber Cell Differentiation and Novel Insights Into Their Denucleation Process
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The temporal and spatial regulation of gene transcription is a central process governing mammalian development and differentiation. Cellular differentiation is executed through coordinated expression of multiple genes, including families of genes encoding proteins with highly specialized functions. Nascent transcription is not a constitutive event as genes are transcribed in sporadic pulses of activity termed transcriptional bursts. There are two transcriptional bursting parameters that regulate output of gene products, burst fraction and burst size/intensity. Transcriptional burst fraction refers to the number of active phases of transcription. Transcriptional burst size refers to the number of RNA molecules produced within a single burst event. It is not known how these parameters change during cell differentiation. Many protein-coding and non-coding genes can be located in gene-rich regions and their nascent transcription can be influenced by promoter location and orientation. Although transcription-coupled splicing generates mRNAs that leave the nuclei, recent studies indicate that specific spliced MRNAs can temporally accumulate in the nuclei. Lens specific transcription of α-, β-, and γ-crystallin genes is a unique system for studies of nascent transcription as the spatial organization of lens fiber cells within the lens correlates precisely with their differentiation status. In addition, lens transparency requires degradation of nuclei in mature lens fibers and this process remains poorly understood. Depletion of ATP dependent chromatin remodeling enzyme Snf2h in lens results in nuclear retention; nevertheless, how Snf2h influences nascent transcription and denucleation cascade remains unknown. My hypothesis predicts that a) Cellular differentiation impacts both transcriptional burst fraction and burst intensity and that individual crystallin genes employ these parameters differently, b) During lens fiber cell differentiation, condensation of chromatin and nuclear compaction prior the denucleation causes "premature" termination of nascent transcription, c) Disruption of lens differentiation by lensspecific depletion of Snf2h impairs transcription of differentiation-regulated genes as the nuclear degradation process, a key part of the differentiation cascade, is impaired, and d), Bi-directional nascent transcription from two crystallin promoters is a rare event due to the local competition for the RNA polymerase II convoys. Specific Aims: 1. To determine transcriptional burst parameters and their changes during lens fiber cell differentiation. 2. To examine the cellular and molecular mechanisms of lens fiber cell denucleation, including cessation of transcription in condensed nuclei and the role of ATPdependent chromatin remodeling enzyme Snf2h (Smarca5) in this process. 3. To investigate transcription from bi-directional promoters and from crystallin generich region of mouse chromosome 5. To study how transcriptional burst fraction and intensity of crystallin genes change during lens development and differentiation (Aim I) we performed RNA fluorescent in situ hybridization (RNA FISH) of αA-, βB1-,βB3-, βA4-, and γA-crystallins and β-actin in the lenses of embryonic day E12.5, E14.5, and E16.5 mouse embryos and newborns. RNA FISH detects nascent transcription sites to visualize active sites of transcription, and, thus, is used to quantify bursting parameters. Lens fiber cells were divided into four different areas, a, b, c, d from the periphery to the center of the lens, respectively, to represent successive differentiation stages. Burst fraction was quantified as percent alleles of a specific crystallin or the β-actin gene transcribed within a given region of the lens. We found that burst fraction changes dramatically during cellular differentiation throughout the lens fiber cell compartment. During early embryonic stages of lens development there was a general increase in burst fraction as cell differentiation progressed. Although burst intensity of crystallin genes also change during lens differentiation and denucleation, this change is moderate compared to the burst fraction parameter. We found unexpectedly that nascent transcription of βB I- and γA-crystallins and β-actin is retained in nuclei just prior their physical disintegration (Aim 2). Nuclear degradation is first marked by changes of the nuclear shape (from ovoid to round), reduction in its size and chromatin condensation. We found that these condensed nuclei transfer both histone and non-histone proteins, including Snf2h, from the nuclei into the cytoplasm. Lens-specific depletion of Snf2h disrupts the denucleation process in the lens; however, the persisting nuclei were still able to transcribe many crystallin genes. Disruption of this chromatin remodeler changed both burst intensity and fraction depending on the crystallin gene being studied (Aim 2). Our data show that nascent transcription from βA4- and βB 1-crystallin promoters can occur frequently from the same allele and that ~β3-crystallin spliced mRNAs are temporally retained within the nuclei of early lens fiber cells (Aim 3).