formaldehyde-assisted-isolation-regulatory-elements-to-measure

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

Justus-Liebig-University

The serine/threonine kinase HIPK2 functions as a regulator of developmental processes and as a signal integrator of a wide variety of stress signals, such as DNA damage, hypoxia, and reactive oxygen intermediates. Because the kinase is generated in a constitutively active form, its expression levels are restricted by a variety of different mechanisms. Here we identify the CCR4-NOT complex as a new regulator of HIPK2 abundance. Down-regulation or knockout of the CCR4-NOT complex member CNOT2 leads to reduced HIPK2 protein levels without affecting the expression level of HIPK1 or HIPK3. A fraction of all HIPK family members associates with the CCR4-NOT components CNOT2 and CNOT3. HIPKs also phosphorylate the CCR4-NOT complex, a feature that is shared with their yeast progenitor kinase, YAK1. Functional assays reveal that HIPK2 and HIPK1 restrict CNOT2-dependent mRNA decay. HIPKs are well known regulators of transcription, but the mutual regulation between CCR4-NOT and HIPKs extends the regulatory potential of these kinases by enabling posttranscriptional gene regulation.

HIPK family kinases bind and regulate the function of the CCR4-NOT complex.

The multi-subunit CCR4 (carbon catabolite repressor 4)-NOT (Negative on TATA) complex serves as a central coordinator of all different steps of eukaryotic gene expression. Here we performed a systematic and comparative analysis of cells where the CCR4-NOT subunits CNOT1, CNOT2 or CNOT3 were individually downregulated using doxycycline-inducible shRNAs. Microarray experiments showed that downregulation of either CNOT subunit resulted in elevated expression of major histocompatibility complex class II (MHC II) genes which are found in a gene cluster on chromosome 6. Increased expression of MHC II genes after knock-down or knock-out of either CNOT subunit was seen in a variety of cell systems and also in naïve macrophages from CNOT3 conditional knock-out mice. CNOT2-mediated repression of MHC II genes occurred also in the absence of the master regulator class II transactivator (CIITA) and did not cause detectable changes of the chromatin structure at the chromosomal MHC II locus. CNOT2 downregulation resulted in an increased de novo transcription of mRNAs whereas tethering of CNOT2 to a regulatory region governing MHC II expression resulted in diminished transcription. These results expand the known repertoire of CCR4-NOT members for immune regulation and identify CNOT proteins as a novel group of corepressors restricting class II expression.

Article	Year
HIPK family kinases bind and regulate the function of the CCR4-NOT complex. Molecular biology of the cell\| PubMed ID: 27122605	2016
The CCR4-NOT complex contributes to repression of Major Histocompatibility Complex class II transcription. Scientific reports\| PubMed ID: 28615693	2017

Olesja Ritter

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