The Sin3-Associated Protein 30 (SAP30) Family of Transcriptional Regulators

Abstract

Elimistön uusi SAP30-proteiiniperhe.

Molekyylitason innovaatiosta sairauksien uusiin hoitomuotoihin?

Epigeneettisellä periytymisellä tarkoitetaan perinnöllisen tiedon siirtoa solun tai eliön jälkeläisille ilman, että perimäaines itsessään, DNA, olisi muuntunut. Molekyylitasolla epigeneettinen periytyminen selittyy erilaisilla geenien luennan toiminnoilla. Tampereen yliopiston lääketieteen laitoksella, FM Keijo Viirin väitöskirjatyössä, löydettiin biologiassa ja lääketieteessä aikaisemmin tuntematon geeni ja sen tuote, toimiva proteiini SAP30L, jonka osoitettiin osallistuvan epigeneettisen periytymisen tapahtumaketjuun. Uuden proteiinin todettiin tehokkaasti vaimentavan perimäaineksen toimintaa.

Jatkotyöt osoittivat geenien vaimennuksen johtuvan tämän uuden SAP30 proteiiniperheen jäsenten ns. sinkkisormirakenteesta, joka sitoi DNA:ta ja taivutti sitä. Uuden proteiiniperheen rakennetta ja toimintaa edelleen selvitettäessä ilmeni, että solujen tuman rasvamolekyylit, tietyt fosfoinositidit, säätelevät SAP30 proteiiniperheen valkuaisaineiden sitoutumista tuman perimäainekseen. Tämän seurauksena SAP30-proteiinit irtoavat perimäaineksesta, jolloin geenien luenta taas tapahtuu. Kyseisiin tässä väitöskirjatyössä selvitettyihin molekyylitason tapahtumiin osallistuivat monet eri tuman valkuaisaineet.

Lääketieteen uusi havaintomme visioidaan muodostavan pohjan aikaisemmin tuntemattomien strategioiden kehittämiselle, joissa estetään sairautta aiheuttavien proteiinien ja perimäaineksen interaktioita. Väitöskirjatyön kyseisestä havainnosta on myös jätetty patenttianomus.

BACKGROUND: The transcription of genes is influenced by their packing status around the nucleosome: tightly packed DNA is inaccessible for RNA polymerase enzymes whereas loosely packed DNA is more efficiently transcribed. The most appreciated histone modification governing the DNA packing is histone tail acetylation and deacetylation. Deacetylation of histones plays a fundamental role in gene silencing by producing deacetylated nucleosomes, where DNA is wrapped more tightly. Deacetylation is often mediated by a corepressor complex (consisting of seven to eight proteins) containing SWI-independent 3A (Sin3A) as an essential scaffold protein. Sin3-Associated Protein 30 (SAP30), has previously been suggested to function as a linker molecule between various corepressors. However, the domain structure and the molecular function of SAP30 protein have remained unknown. AIMS: The purpose of this study was to identify regulatory proteins in the differentiating intestinal epithelial cells and to further decipher their function in closer molecular detail.

RESULTS: We identified a novel transforming growth factor-beta (TGF-beta)-upregulated mRNA species in a mesenchymal-epithelial cell co-culture model which mimics the intestinal crypt villus axis biology in terms of epithelial cell differentiation. mRNA was ubiquitously expressed in various tissues and encoded a nuclear localizing protein with 70% identity with SAP30 and we thus named it as SAP30-like (SAP30L) (I). SAP30L interacts with several components of the Sin3A corepressor complex and binds to the PAH3/HID (Paired Amphipathic Helix 3/Histone deacetylase Interacting Domain) region of Sin3A. Like SAP30, when tethered to promoters, SAP30L induces strong transcriptional repression in a manner dependent on Sin3A and histone deacetylases. We discovered a functional nucleolar localization signal in SAP30L and showed that SAP30L and SAP30 (hereafter called SAP30 proteins) are able to target Sin3A to the nucleolus (II). In further structure-function mapping of the SAP30 proteins we identified a zinc-coordinating structure which is necessary for DNA binding and found that one consequence of binding is bending of the DNA. We also showed that nuclear signaling lipids, phosphoinositides (PIs), bind to the same region in the SAP30 proteins as DNA. In fact, PIs and DNA evince an antagonizing interrelationship in regard to their binding to the SAP30 proteins, since binding of PIs detaches SAP30 proteins from the chromatin. PI binding also reduces the repressive activity of the SAP30 proteins and affects their translocation from the nucleus to the cytoplasm. In addition, the repressional activity of the SAP30 proteins is partly dependent on their direct interaction with the globular domain of the core histones and nucleosomes (III). Our molecular evolutionary analysis indicated that SAP30L is the ancestral protein of the SAP30 protein family and that SAP30 originated by a chromosome segment duplication which occurred after the divergence of Actinopterygii (ray-finned fishes) and Sarcopterygii (flesh-finned fishes) about 4 50 million years ago. Phylogenetic analysis and biochemical experiments suggested that SAP30 has diverged functionally from the ancestral SAP30L by accumulating mutations causing attenuation of one of the original functions, association with the nuclear matrix (IV).

CONCLUSIONS: First, we identified a novel epigenetic regulator protein, SAP30L, in differentiating human intestinal epithelial cells. In general, SAP30L might have a role in recruiting the Sin3-histone deacetylase complex to specific corepressor subcomplexes in response to TGF-beta, thus leading to the silencing of proliferation-driving genes in these differentiating intestinal epithelial cells. Our detailed results suggest that SAP30L and SAP30 mediate gene repression through multiple interactions, i.e. with Sin3A, histone deacetylases (HDACs), DNA and histones/nucleosomes.

Furthermore, the ability of the SAP30 family proteins to bend DNA suggests that they are active partners in complexes regulating gene expression. This work describes multifarious interactions of SAP30 proteins in the promoter-bound Sin3 repressome which can be utilized in drug design to destabilize SAP30-containing Sin3 complexes in diseases wherever they are presumably implicated (e.g. cancer and viral infections). Moreover, the findings here shed light on an ambiguous, already half-century ago characterized, nuclear phospholipid component of the cell by proposing that nuclear signaling lipids, phosphoinositides (PIs), regulate protein-DNA interactions. Thus, part of the PI signaling-mediated changes in gene expression are probably due to the ability of certain proteins (such as the SAP30 proteins) to sense the nuclear PI content and to detach from chromatin when a threshold concentration of PI is reached. Finally, PI-mediated inhibition of protein-DNA interactions is discussed in terms of its potential as a novel strategy for inhibiting pathogenic protein-chromatin interactions and its utilization in combinatorial therapies with e.g. HDAC inhibitors.