Mechanisms controlling SMAD-dependent transcription and chromatin modification

Abstract

Regulation of molecular processes in the cell is critical for proper cellular function and identity. To maintain this function and identity, proteins derived from approximately 20,000 protein coding genes encoded on the human genome need to be present at the right place and at the right time. Eukaryotic cells possess various strategies to control the activity and spatiotemporal distribution of proteins at the transcriptional and post-transcriptional levels. The human reference genome roughly translates to 2 m of DNA per diploid genome that needs to be fit inside its 6 µm diameter cell nucleus. To efficiently store and access the genetic information in the nucleus the DNA is wrapped around an octamer of core histone proteins forming nucleosomes. The histone tails that protrude from the nucleosome contain several residues that can become modified by addition and removal of post-translational modifications (PTMs) by multiprotein complexes like the Mixed Lineage Leukemia (MLL) methyltransferase, Polycomb Repressive Deubiquitinase (PR-DUB) and Mitotic Deacetylase (MiDAC) complexes. The nature and locations of these PTMs define the chromatin environment and control on a transcriptional level whether a gene is transcribed or silenced. SMA and MAD (SMAD) gene specific transcription factors (GSTFs) relay cellular signals induced by the Transforming Growth Factor-β (TGF-β) and the Bone Morphogenetic Protein (BMP) cytokines. Activation of the TGF-β receptor induces phosphorylation of the TGF-β Receptor-SMADs (R-SMADs) SMAD2/3, while activation by BMP induces the R-SMADs SMAD1/5/9. SMADs require active protein transport between cytoplasm and nucleus for proper functioning that is mediated by transport proteins. Once the SMADs are in the nucleus they can bind to their respective DNA targets. By constraining these SMAD GSTFs in the cytoplasm the activity of these transcription factors is controlled by modulation of their nuclear import. This thesis aims to investigate various control mechanisms on transcriptional and post-transcriptional levels. In this thesis we describe Chromatin Regulation Ontology SiRNA Screening (CROSS), a microscopy-based high-throughput screening method that allows for identification of proteins influencing histone PTM levels. By combining siRNA knockdown with automated antibody-based staining and automated microscopy we show CROSS can be used to identify writers and erasers of chromatin marks. Additionally, we describe identification of IPO5 via affinity purification coupled with mass spectrometry as a de novo binding partner of BMP-activated SMAD1. Using confocal microscopy we show that IPO5 is a BMP-activated SMAD specific transporter and that the lysine stretch found in the nuclear localization signal of the R-SMADs is a determinant for IPO specificity. Next, we describe the involvement of the MLL4 methyltransferase complex in TGF-β induced SMAD signaling. Direct interaction of MLL3/4 subunits PA1 and PTIP is shown for SMAD2/3/4. Expression analysis of TGF-β regulated genes shows that their activation by TGF-β is dependent on the MLL4 complex. Finally, we explore the interactome composition of the MLL3/MLL4 complex subunit UTX. By mass spectrometric analysis we confirm the interaction with MLL3/4 and PR-DUB. Additionally, we identify UTX to be part of the MiDAC complex.