ZNF410 represses fetal globin by devoted control of CHD4/NuRD [preprint]

Major effectors of adult-stage fetal globin silencing include the transcription factors (TFs) BCL11A and ZBTB7A/LRF and the NuRD chromatin complex, although each has potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here through CRISPR screening we discover ZNF410 to be a novel fetal hemoglobin (HbF) repressing TF. ZNF410 does not bind directly to the γ-globin genes but rather its chromatin occupancy is solely concentrated at CHD4, encoding the NuRD nucleosome remodeler, itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for ZNF410’s effects on γ-globin repression. Knockout of ZNF410 reduces CHD4 by 60%, enough to substantially de-repress HbF while avoiding the cellular toxicity of complete CHD4 loss. Mice with constitutive deficiency of the homolog Zfp410 are born at expected Mendelian ratios with unremarkable hematology. ZNF410 is dispensable for human hematopoietic engraftment potential and erythroid maturation unlike known HbF repressors. These studies identify a new rational target for HbF induction for the β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved transcription factor with singular devotion to regulation of a chromatin subcomplex

Inhibition of Smad -mediated activin and TGFbeta signaling by calmodulin.

Smad proteins are a family of transcriptional regulators that mediate signaling by activin, transforming growth factor beta, and related ligands. Ligand binding to specific heteromeric receptor serine kinase complexes at the cell surface results in phosphorylation of receptor-regulated Smads, subsequent association with a common Smad, and nuclear translocation of the complex. Therefore, Smads translate TGFbetaP signals directly into changes in gene expression. Analysis of Smad function in Xenopus indicates that Smad2 functions in activin-specific pathways, while Smad1 mediates BMP-specific signals. The importance of this family of proteins is highlighted by the observation that loss of Smad2 or Smad4 function is associated with tumor formation in humans. In order to identify proteins which functionally interact with Smad2, we screened a bacteriophage expression library of 16-day mouse embryo cDNA using radiolabelled Smad2 as probe. Screening of 1.5 x 106 total plaques resulted in the isolation of seven cDNAs encoding Smad2 binding proteins. All seven were subsequently sequenced and determined to be calmodulin. Calmodulin is the primary intracellular receptor for calcium ions and, when saturated with calcium, is known to bind basic amphiphilic alpha-helices in target proteins. Smad2 protein specifically associates with calmodulin-agarose in a calcium-dependent manner in vitro. Calmodulin binds to two different basic amphiphilic alpha-helices in Smad2; the affinity constant for the most N-terminal binding helix is 30 nM, while the second target helix binds calmodulin with muM affinity. In the same assay, Smads 1, 3, and 4 also associate with calmodulin-agarose. Increasing the amount of active calmodulin in cultured cells by overexpression inhibits TGFbeta and Smad-dependent transcriptional activation in a dose-dependent manner. Elevation of intracellular calcium by ionomycin has the same dose-dependent inhibitory effect, while a calmodulin antagonist, W13, increases reporter gene expression. In addition, exogenous calmodulin inhibits Smad-initiated transcriptional activation in the absence of functional TGFbeta type I receptor demonstrating that inhibition occurs at the level of Smad function. Finally, calmodulin blocks Smad2-induced mesoderm formation in Xenopus embryos. These observations demonstrate that calmodulin can bind to two different alpha-helices within receptor-regulated Smad proteins with physiological affinity, and can inhibit Smad-mediated signaling in cell culture and in vivo.Ph.D.BiochemistryBiological SciencesMolecular biologyPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132285/2/9959897.pd

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written several papers on livestock management in wilderness

Response of mountain meadows to grazing by recreational pack stock

Effects of recreational pack stock grazing on mountain meadows in Yosemite National Park were assessed in a 5-year study. Yosemite is a designated wilderness, to be managed such that its natural conditions are preserved. Studies were conducted in 3 characteristic meadow types: shorthair sedge (Carex filifolia Nutt.), Brewer's reed grass (Calamagrostis breweri Thurber), and tufted hairgrass [Deschampsia cespitosa (L.) Beauv.]. Horses and mules grazed experimental plots at intensities of 15 to 69% utilization for 4 seasons. In all 3 meadows, grazing caused decreases in productivity. The mean reduction after 4 years of grazing was 18% in the shorthair sedge meadow, 17% in the Brewer's reed grass meadow, and 22% in the tufted hairgrass meadow. Grazing also caused shifts in basal groundcover (usually a reduction in vegetation cover and increase in bare soil cover), and changes in species composition. Productivity and vegetation cover decreased as percent utilization increased, while bare soil cover increased as utilization increased. Changes in species composition were less predictably related to differences in grazing intensity. Passive management of grazing is insufficient in wilderness areas that are regularly used by groups with recreational stock. Wilderness managers need to monitor meadow conditions and the grazing intensities that occur. Our study suggests that biomass and ground cover are more sensitive indicators of grazing impact than species composition. Managers must make decisions about maximum acceptable levels of grazing impact and then develop guidelines for maximum use levels, based on data such as ours that relates grazing intensity to meadow response.The Journal of Range Management archives are made available by the Society for Range Management and the University of Arizona Libraries. Contact [email protected] for further information.Migrated from OJS platform August 202

CRISPRO: identification of functional protein coding sequences based on genome editing dense mutagenesis

Abstract CRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets, we develop CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genomes, transcripts, and protein coordinates and structures. No currently available tool has similar functionality. The ensuing genotype-phenotype linear and three-dimensional maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro

Distinct nuclear compartment-associated genome architecture in the developing mammalian brain

Nuclear compartments are thought to play a role in three-dimensional genome organization and gene expression. In mammalian brain, the architecture and dynamics of nuclear compartment-associated genome organization is not known. In this study, we developed Genome Organization using CUT and RUN Technology (GO-CaRT) to map genomic interactions with two nuclear compartments-the nuclear lamina and nuclear speckles-from different regions of the developing mouse, macaque and human brain. Lamina-associated domain (LAD) architecture in cells in vivo is distinct from that of cultured cells, including major differences in LADs previously considered to be cell type invariant. In the mouse and human forebrain, dorsal and ventral neural precursor cells have differences in LAD architecture that correspond to their regional identity. LADs in the human and mouse cortex contain transcriptionally highly active sub-domains characterized by broad depletion of histone-3-lysine-9 dimethylation. Evolutionarily conserved LADs in human, macaque and mouse brain are enriched for transcriptionally active neural genes associated with synapse function. By integrating GO-CaRT maps with genome-wide association study data, we found speckle-associated domains to be enriched for schizophrenia risk loci, indicating a physical relationship between these disease-associated genetic variants and a specific nuclear structure. Our work provides a framework for understanding the relationship between distinct nuclear compartments and genome function in brain development and disease