Regulation of nuclear actin levels and MRTF/SRF target gene expression during PC6.3 cell differentiation

Actin has important functions in both cytoplasm and nucleus of the cell, with active nuclear transport mechanisms maintaining the cellular actin balance. Nuclear actin levels are subject to regulation during many cellular processes from cell differentiation to cancer. Here we show that nuclear actin levels increase upon dif-ferentiation of PC6.3 cells towards neuron-like cells. Photobleaching experiments demonstrate that this increase is due to decreased nuclear export of actin during cell differentiation. Increased nuclear actin levels lead to decreased nuclear localization of MRTF-A, a well-established transcription cofactor of SRF. In line with MRTF-A localization, transcriptomics analysis reveals that MRTF/SRF target gene expression is first transiently activated, but then substantially downregulated during PC6.3 cell differentiation. This study therefore describes a novel cellular context, where regulation of nuclear actin is utilized to tune MRTF/SRF target gene expression during cell differentiation.Peer reviewe

Lamina-associated polypeptide 2 alpha is required for intranuclear MRTF-A activity

Myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), regulates the expression of many cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Here we describe a novel mechanism to regulate MRTF-A activity within the nucleus by showing that lamina-associated polypeptide 2 alpha (Lap2 alpha), the nucleoplasmic isoform of Lap2, is a direct binding partner of MRTF-A, and required for the efficient expression of MRTF-A/SRF target genes. Mechanistically, Lap2 alpha is not required for MRTF-A nuclear localization, unlike most other MRTF-A regulators, but is required for efficient recruitment of MRTF-A to its target genes. This regulatory step takes place prior to MRTF-A chromatin binding, because Lap2 alpha neither interacts with, nor specifically influences active histone marks on MRTF-A/SRF target genes. Phenotypically, Lap2 alpha is required for serum-induced cell migration, and deregulated MRTF-A activity may also contribute to muscle and proliferation phenotypes associated with loss of Lap2 alpha. Our studies therefore add another regulatory layer to the control of MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2 alpha in transcriptional regulation.Peer reviewe

RNA export factor Ddx19 is required for nuclear import of the SRF coactivator MKL1

Controlled transport of macromolecules between the cytoplasm and nucleus is essential for homeostatic regulation of cellular functions. For instance, gene expression entails coordinated nuclear import of transcriptional regulators to activate transcription and nuclear export of the resulting messenger RNAs for cytoplasmic translation. Here we link these two processes by reporting a novel role for the mRNA export factor Ddx19/Dbp5 in nuclear import of MKL1, the signal-responsive transcriptional activator of SRF. We show that Ddx19 is not a general nuclear import factor, and that its specific effect on MKL1 nuclear import is separate from its role in mRNA export. Both helicase and nuclear pore-binding activities of Ddx19 are dispensable for MKL1 nuclear import, but RNA binding is required. Mechanistically, Ddx19 operates by modulating the conformation of MKL1, which affects its interaction with Importin-beta for efficient nuclear import. Thus, Ddx19 participates in mRNA export, translation and nuclear import of a key transcriptional regulator.Peer reviewe

Nuclear actin dynamics in gene expression and genome organization

Although best known from its functions in the cytoplasm, actin also localizes to the cell nucleus, where it has been linked to many essential functions from regulation of gene expression to maintenance of genomic integrity. While majority of cytoplasmic functions of actin depend on controlled actin polymerization, in the nucleus both actin monomers and filaments have their own specific roles. Actin monomers are core components of several chromatin remodeling and modifying complexes and can also regulate the activity of specific transcription factors, while actin filaments have been linked to DNA damage response and cell cycle progression. Consequently the balance between monomeric and filamentous actin must be precise controlled also in the nucleus, since their effects are dynamically coupled. In this review, we discuss the recent data on how actin dynamics is regulated within the nucleus and how this influences the different nuclear processes dependent on actin.Peer reviewe

Biochemical Characterization of Nuclear Actin Interactions

Aktiini tunnetaan yleisenä soluliman proteiinina, jonka tärkeimpiä tehtäviä ovat muun muassa solun tukirangan muodostus ja osallistuminen liikkeen tuottamiseen solussa yhdessä myosiinien kanssa. Aktiinia esiintyy kuitenkin myös tumassa, jossa sen on osoitettu osallistuvan geenien ilmentymisen säätelyyn sekä itsenäisesti että osana kromatiininmuokkauskomplekseja. Tärkeänä osana tuman aktiinin tutkimusta on selvittää siihen sitoutuvia muita tuman proteiineja. Näiden interaktioiden kautta aktiini voidaan yhdistää uusiin toimintoihin tumassa. Tämän maisterintutkielman tavoitteena on ollut tuottaa ja puhdistaa viittä eri tuman proteiinia, joiden on ehdotettu sitoutuvan aktiiniin ja tutkia puhdistettujen proteiinien sitoutumista aktiiniin sen monomeeri- ja filamenttimuodoissa. Tutkittavat proteiinit olivat aktiinin kaltaiset (actin-related protein: ARP) proteiinit Arp4, Arp5, Arp6 ja Arp8 sekä RNA polymeraasi II:n alayksikkö Rpb8. Tutkimukseen valituista proteiineista Arp4:n ja Rpb8:n tuotto ja puhdistus onnistuivat. Näistä proteiineista kumpikaan ei sitoutunut monomeeriseen aktiiniin korkealla affiniteetilla, mutta Rpb8 vaikutti sitovan aktiinisäikeitä. Aktiinin polymerisaatioon Rpb8 ei sen sijaan vaikuttanut. Tulokset tuovat uusia näkökantoja aiheeseen, sillä aikaisemmin on ajateltu Arp4:n muodostavan aktiinin kanssa heterokompleksin, mitä tämän tutkielman tulokset eivät tue. Rpb8:n sitoutumisesta tuman aktiiniin osana RNA polymeraasi II:sta oli puolestaan aikaisemmin niukasti tietoa, joten tulokset tuovat uutta tietoa aktiinin osuudesta polymeraasin toiminnassa.Actin is known as abundant cytoplasmic protein, which functions as a component of the cytoskeleton and in cell motility together with motor protein myosins. However, actin is also present in the nucleus, where it has been shown to take part in the control of gene expression, both independently and as part of chromatin remodeling complexes. An important aspect in the study of nuclear actin is to identify other nuclear proteins interacting with actin and to confirm these interactions in biochemical experiments. Through these interactions actin can be linked to various nuclear processes. The aim of this master’s thesis study was to express and purify five nuclear proteins that have been suggested to bind actin and to study the binding in detail both with actin filaments and monomers. The proteins of interest include four actin-related proteins (ARPs) Arp4, Arp5, Arp6 and Arp8 as well as RNA polymerase II subunit Rpb8. Out of the proteins selected for this study, the expression and purification of Arp4 and Rpb8 was successful. Neither one did bind monomeric actin with high affinity, but interestingly Rpb8 did bind actin filaments. On the other hand, Rpb8 did not have any effect on actin polymerization. These results provide new insights into nuclear actin function. It has been suggested earlier that Arp4 would form a heterocomplex with actin, but the results of this study do not support this. The binding between Rpb8 and actin in RNA polymerase II complex has not been extensively studied before, so the results provide new information about the function of actin in the polymerase complex