S-Phase Progression Mediates Activation of a Silenced Gene in Synthetic Nuclei

Abstract

Aberrant expression of developmentally silenced genes, characteristic of tumor cells and regenerating tissue, is highly correlated with increased cell proliferation. By modeling this process in vitro in synthetic nuclei, we find that DNA replication leads to deregulation of established developmental expression patterns. Chromatin assembly in the presence of adult mouse liver nuclear extract mediates developmental stage-specific silencing of the tumor marker gene alpha-fetoprotein (AFP). Replication of silenced AFP chromatin in synthetic nuclei depletes sequence-specific transcription repressors, thereby disrupting developmentally regulated repression. Hepatoma-derived factors can target partial derepression of AFP, but full transcription activation requires DNA replication. Thus, unscheduled entry into S phase directly mediates activation of a developmentally silenced gene by (i) depleting developmental stage-specific transcription repressors and (ii) facilitating binding of transactivators.Cellular commitment, differentiation, and specificity are determined primarily by the interaction of protein complexes with chromatin DNA. This gene-regulatory programming is challenged during each cell cycle by passage of the replisome during DNA synthesis. DNA replication may facilitate competition between transiently disrupted histones and transacting factors at the replication fork, potentially changing established chromatin structure and gene expression patterns (2,7,20,29,59). During tumorigenesis, terminally differentiated cells that have withdrawn from the cell cycle are induced to resume cycling, often through functional inactivation of tumor suppressor genes such as Rb and INK4a (30,33). This cyclic disruption of chromatin structure provides a fertile environment, similar to that which exists during fetal development, for altering gene expression patterns. Aberrant adult activation of genes normally expressed only in the fetus is characteristic of many tumors (reviewed in references 31 and 39). For example, a hepatoma marker gene, alpha-fetoprotein (AFP), is transcriptionally repressed shortly after birth (reviewed in reference 52) and is reexpressed in mature hepatocytes only when they leave G 0 quiescence and begin cycling following partial hepatectomy or during hepatocellular carcinoma (HCC) (42; reviewed in reference 52). Expression of AFP is therefore closely linked to cell cycle progression; the renewal of DNA replication following transition from quiescence to S phase may play an epigenetic role in modulating gene expression.The relative local concentration of transacting factors at the time of replication can determine whether a given chromatin structure is maintained or converted to an alternate conformation (17, 58). Modulating the balance of repressors and activators present during replication could be achieved through a variety of mechanisms, including functional inactivation of repressors and activation of transcription factors. Alternatively, the availability of transactivators could be controlled by s...

Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver

“…Postnatal AFP repression in liver is coincident with the cessation of hepatocyte cell division, and AFP reactivation is often related to the hepatocyte proliferation (e.g., regeneration and carcinogenesis) (1). It is thought that there may be a mechanistic link behind this temporal correlation, which is further supported by in vitro studies of AFP reactivation (30). However, the adult LZB20KO liver has a normal architecture and remains in a normally nonproliferating status, despite their aberrant expression of AFP.…”

p53-Targeted LSD1 Functions in Repression of Chromatin Structure and Transcription In Vivo

“…We performed ChIP analyses of developmentally staged mouse liver tissue to determine relative levels of endogenous p53, LSD1, and H3K4me2 present at the developmental repressor region (SBE/p53RE) (60) of AFP chromatin. Our previous work showed that this intercalated Smad/p53 response element is essential for p53-mediated repression of AFP in cultured cells and by in vitro transcription (11,34). Shortly after birth, when hepatic AFP expression is robust (Fig.…”

Cell-Cycle Dependence of Transcription Dominates Noise in Gene Expression

“…At low expression tetO promoters exhibit transcriptional pulses that originate around the G1 to S transition when DNA replication occurs. S phase progression has been suggested to affect transcription [47] and has been established as important to (de)silence gene loci in a manner that either requires replication fork progression [48], [49] or not [50], [51]. Replication-dependent transcriptional activation of viral genes has been reported in transient transfection assays, and depends on trans activators that bind to either the proximal promoter or and enhancer region [52], [53].…”