Dorota Skowronska-Krawczyk

Neurogenin 2 controls cortical neuron migration through regulation of Rnd2

Motility is a universal property of newly generated neurons. How cell migration is coordinately regulated with other aspects of neuron production is not well understood. Here we show that the proneural protein neurogenin 2 (Neurog2), which controls neurogenesis in the embryonic cerebral cortex, directly induces the expression of the small GTP-binding protein Rnd2 (ref. 3) in newly generated mouse cortical neurons before they initiate migration. Rnd2 silencing leads to a defect in radial migration of cortical neurons similar to that observed when the Neurog2 gene is deleted. Remarkably, restoring Rnd2 expression in Neurog2-mutant neurons is sufficient to rescue their ability to migrate. Our results identify Rnd2 as a novel essential regulator of neuronal migration in the cerebral cortex and demonstrate that Rnd2 is a major effector of Neurog2 function in the promotion of migration. Thus, a proneural protein controls the complex cellular behaviour of cell migration through a remarkably direct pathway involving the transcriptional activation of a small GTP-binding protein.

“Seq-ing” Insights into the Epigenetics of Neuronal Gene Regulation

The epigenetic control of neuronal gene expression patterns has emerged as an underlying regulatory mechanism for neuronal function, identity, and plasticity, in which short- to long-lasting adaptation is required to dynamically respond and process external stimuli. To achieve a comprehensive understanding of the physiology and pathology of the brain, it becomes essential to understand the mechanisms that regulate the epigenome and transcriptome in neurons. Here, we review recent advances in the study of regulated neuronal gene expression, which are dramatically expanding as a result of the development of new and powerful contemporary methodologies, based on next-generation sequencing. This flood of new information has already transformed our understanding of many biological processes and is now driving discoveries elucidating the molecular mechanisms of brain function in cognition, behavior, and disease and may also inform the study of neuronal identity, diversity, and neuronal reprogramming.

Highly specific interactions between bHLH transcription factors and chromatin during retina development

Basic helix-loop-helix (bHLH) transcription factors such as atonal homolog 5 (ATH5) and neurogenin 2 (NGN2) determine crucial events in retinogenesis. Using chromatin immunoprecipitation, we demonstrate that their interactions with target promoters undergo dynamic changes as development proceeds in the chick embryo. Chick ATH5 associates with its own promoter and with the promoter of the β3 nicotinic receptor specifically in retinal ganglion cells and their precursors. NGN2 binds to the ATH5 promoter in retina but not in optic tectum, suggesting that interactions between bHLH factors and chromatin are highly tissue specific. The transcriptional activations of both promoters correlate with dimethylation of lysine 4 on histone H3. Inactivation of the ATH5 promoter in differentiated neurons is accompanied by replication-independent chromatin de-methylation. This report is one of the first demonstrations of correlation between gene expression, binding of transcription factors and chromatin modification in a developing neural tissue.

Lens regeneration using endogenous stem cells with gain of visual function

The repair and regeneration of tissues using endogenous stem cells represents an ultimate goal in regenerative medicine. To our knowledge, human lens regeneration has not yet been demonstrated. Currently, the only treatment for cataracts, the leading cause of blindness worldwide, is to extract the cataractous lens and implant an artificial intraocular lens. However, this procedure poses notable risks of complications. Here we isolate lens epithelial stem/progenitor cells (LECs) in mammals and show that Pax6 and Bmi1 are required for LEC renewal. We design a surgical method of cataract removal that preserves endogenous LECs and achieves functional lens regeneration in rabbits and macaques, as well as in human infants with cataracts. Our method differs conceptually from current practice, as it preserves endogenous LECs and their natural environment maximally, and regenerates lenses with visual function. Our approach demonstrates a novel treatment strategy for cataracts and provides a new paradigm for tissue regeneration using endogenous stem cells.

Required enhancer-matrin-3 network interactions for a homeodomain transcription program

Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) and β-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with β-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with β-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

Conserved regulatory sequences in Atoh7 mediate non- conserved regulatory responses in retina ontogenesis

The characterisation of interspecies differences in gene regulation is crucial to understanding the molecular basis of phenotypic diversity and evolution. The atonal homologue Atoh7 participates in the ontogenesis of the vertebrate retina. Our study reveals how evolutionarily conserved, non-coding DNA sequences mediate both the conserved and the species-specific transcriptional features of the Atoh7 gene. In the mouse and chick retina, species-related variations in the chromatin-binding profiles of bHLH transcription factors correlate with distinct features of the Atoh7 promoters and underlie variations in the transcriptional rates of the Atoh7 genes. The different expression kinetics of the Atoh7 genes generate differences in the expression patterns of a set of genes that are regulated by Atoh7 in a dose-dependent manner, including those involved in neurite outgrowth and growth cone migration. In summary, we show how highly conserved regulatory elements are put to use in mediating non-conserved functions and creating interspecies neuronal diversity.

The Basic Domain of ATH5 Mediates Neuron-Specific Promoter Activity during Retina Development

ABSTRACT In the developing retina, the gene encoding the β3 subunit of the neuronal nicotinic receptor, a specific marker of retinal ganglion cells, is under the direct control of the atonal homolog 5 (ATH5) basic helix-loop-helix (bHLH) transcription factor. Although quite short (143 bp in length), the β3 promoter has the remarkable capacity to discriminate between ATH5 and the other neuronal bHLH proteins expressed in the developing nervous system. We have identified three amino acids within the basic domain that confer specificity to the ATH5 protein. These residues do not mediate direct DNA binding but are required for interaction between ATH5 and chromatin-associated proteins during retina development. When misexpressed in neurons, the myogenic bHLH factor MyoD is also able to activate the β3 gene. This, however, is achieved not by binding of the protein to the promoter but by dimerization of MyoD with a partner, a process that depends not on the basic domain but on the HLH domain. By sequestering an E-box-binding protein, MyoD relieves the active repression that blocks the β3 promoter in most neurons. The mechanisms used by bHLH proteins to activate β3 thus highlight how ATH5 is selected by the β3 promoter and coordinates the derepression and transcriptional activation of the β3 gene during the specification of retinal ganglion cells.

P16INK4a Upregulation Mediated by SIX6 Defines Retinal Ganglion Cell Pathogenesis in Glaucoma

Glaucoma, a blinding neurodegenerative disease, whose risk factors include elevated intraocular pressure (IOP), age, and genetics, is characterized by accelerated and progressive retinal ganglion cell (RGC) death. Despite decades of research, the mechanism of RGC death in glaucoma is still unknown. Here, we demonstrate that the genetic effect of the SIX6 risk variant (rs33912345, His141Asn) is enhanced by another major POAG risk gene, p16INK4a (cyclin-dependentxa0kinase inhibitor 2A, isoform INK4a). Wexa0further show that the upregulation of homozygous SIX6 risk alleles (CC) leads to an increase in p16INK4a expression, with subsequent cellular senescence, as evidenced in a mouse model of elevated IOP and inxa0human POAG eyes. Our data indicate that SIX6 and/or IOP promotes POAG by directly increasing p16INK4a expression, leading to RGC senescence in adult human retinas. Our study provides important insights linking genetic susceptibility to the underlying mechanism of RGC death and provides a unified theory of glaucoma pathogenesis.

A Positive Feedback Loop between ATOH7 and a Notch Effector Regulates Cell-Cycle Progression and Neurogenesis in the Retina

The HES proteins are known Notch effectors and have long been recognized as important in inhibiting neuronal differentiation. However, the roles that they play in the specification of neuronal fate remain largely unknown. Here, we show that in the differentiating retinal epithelium, the proneural protein ATOH7 (ATH5) is required for the activation of the transcription of the Hes5.3 gene before the penultimate mitosis of progenitor cells. We further show that the HES5.3 protein slows down the cell-cycle progression of Atoh7-expressing cells, thereby establishing conditions for Atoh7 to reach a high level of expression in S phase and induce neuronal differentiation prior to the ultimate mitosis. Our study uncovers how a proneural protein recruits a protein known to be a component of the Notch signaling pathway in order to regulate the transition between an initial phase of selection among uncommitted progenitors and a later phase committing the selected progenitors to neuronal differentiation.

Highly Conserved Sequences Mediate the Dynamic Interplay of Basic Helix-Loop-Helix Proteins Regulating Retinogenesis *

The atonal homolog 5 (ATH5) protein is central to the transcriptional network regulating the specification of retinal ganglion cells, and its expression comes under the spatiotemporal control of several basic helix-loop-helix (bHLH) proteins in the course of retina development. Monitoring the in vivo occupancy of the ATH5 promoter by the ATH5, Ngn2, and NeuroM proteins and analyzing the DNA motifs they bind, we show that three evolutionarily conserved E-boxes are required for the bHLH proteins to control the different phases of ATH5 expression. E-box 4 mediates the activity of Ngn2, ATH5, and NeuroM along the pathway leading to the conversion of progenitors into newborn neurons. E-box 1, by mediating the antagonistic effects of Ngn2 and HES1 in proliferating progenitors, controls the expansion of the ATH5 expression domain in early retina. E-box 2 is required for the positive feedback by ATH5 that underlies the up-regulation of ATH5 expression when progenitors are going through their last cell cycle. The combinatorial nature of the regulation of the ATH5 promoter suggests that the bHLH proteins involved have no assigned E-boxes but use a common set at which they either cooperate or compete to finely tune ATH5 expression as development proceeds.