Transcriptional burst kinetics are linked to short term transcriptional memory

Transcriptional bursting is thought to be a stochastic process that allows the dynamic regulation of most genes. The random telegraph model assumes the existence of two states, ON and OFF. However recent studies indicate the presence of additional ON states, suggesting that bursting kinetics and their regulation can be quite complex. We have developed a system to study transcriptional bursting in the context of p53 biology using the endogenous p21 gene tagged with MS2 in human cells. Remarkably, we find that transcriptional bursts from the p21 gene contain multiple ON and OFF states that can be regulated by elevation of p53 levels. Distinct ON states are characterized by differences in burst duration, classified as Short and Long, with long bursts associated with higher Pol II initiation rates. Importantly, the different ON states display memory effects that allow us to predict the likelihood of properties of future bursting events. Long bursting events result in faster re-activation, longer subsequent bursts and higher transcriptional output in the future compared to short bursts. Bursting memory persists up to 2 hours suggesting a stable inheritable promoter architecture. Bursting memory at the p21 gene is the strongest under basal conditions and is suppressed by UV and inhibition of H3K9me1/2, which also increase transcriptional noise. Stabilization of p53 by Nutlin-3a partially reverses suppression of bursting memory suggesting that higher p53 levels may be a key in enforcing memory under conditions of cellular stress. Overall our data uncover a new found bursting property termed Short-Term Transcriptional Memory (STTM) that has the potential to fine-tune transcriptional output at the p21 gene.

Clustered PHD domains in mixed lineage leukaemia proteins are attracted by acetylation-rich active promoters and enhancers

Histone lysine methyltransferase (KMT2) proteins form the core of COMPASS and COMPASS-like complexes that mediate transcriptional memory by methylating H3K4 at promoters and enhancers. KMT2A-D proteins, alternatively called mixed lineage leukaemia proteins (MLL1-4), contain highly conserved unique triplet and quartet of plant homeodomains (PHDs). Here, we show that clustered PHDs, expressed in isolation in HeLa cells, localize to well-defined loci of acetylation-rich active promoters and enhancers. Binding sites overlap with targets of full-length KMT2A (MLL1) and the COMPASS-like subunit WDR5, RbBP5 and with cell cycle and cancer-related genes. COSMIC data identify frequent variations in the PHDs of KMT2 proteins, particularly KMT2C, in a wide spectrum of malignancies. Changes are enriched at conserved positions within the PHDs, indicating that they cause loss-of-function mutations. Taken together, the biochemical and cancer data suggest that the PHDs contribute to KMT2A-D targeting to active promoters and enhancers.

Abstract P347: Transcriptional Features Of Biological Age Maintained In Cultured Cardiac Interstitial Cells

Introduction: Ex vivo expansion of cells is necessary in regenerative medicine to generate large populations for therapeutic use. Adaptation to culture conditions prompt an increase in transcriptome diversity and decreased population heterogeneity in cKit+ cardiac interstitial cells (cCICs). The “transcriptional memory” influenced by cellular origin remains unexplored and is likely to differ between neonatal versus senescent input cells undergoing culture expansion. Approach: cCICs isolated from neonatal and adult cardiac tissue (Left Ventricular Assist Device; LVAD). Single cell libraries from in vitro expanded cells were prepared following five passages as previously demonstrated by our group to promote transcriptional homogeneity. “Transcriptional memory” was surveyed via bioinformatic analysis including unsupervised clustering, differential expression analysis, gene ontology and pathway analysis. Transitional states were assessed through pseudotime analysis. Results: Cell cycle imprint associated with biological age after culture was observed in Neonatal cCICs via upregulation of G2M genes. LVAD derived cCICs retained a widespread senescent profile, in particular high expression of interleukins and elements of the senescence associated secretory phenotype (SASP). Nuclear pore complex TPR and UBC9 and translation initiation factors, displayed age-associated downregulation of elements in the RNA transport and processing pathway. Pathway and co-expression analysis of fibroblast markers Ddr2, Tcf21, Vimentin, Periostin and Collagen deposition markers indicated a primed fibrotic phenotype in senescent cells. A small subset of cCICs exist in a transcriptional continuum between “youthful” phenotype and the damaged microenvironment of adult tissue in which they were embedded. Conclusion: The influence of age, pathology and the cellular stress associated to the in vivo tissue microenvironment persist after culture adaptation, influencing targets of 1) cell cycle, 2) senescence associated secretory phenotype (SASP), 3) RNA transport, and 4) ECM-receptor/fibrosis. The connate transcriptional phenotypes offer fundamental biological insight and highlights cellular input as a consideration in culture expansion and adoptive transfer protocols.

The control of transcriptional memory by stable mitotic bookmarking

To maintain cellular identities during development, gene expression profiles must be faithfully propagated through cell generations. The reestablishment of gene expression patterns upon mitotic exit is thought to be mediated, in part, by mitotic bookmarking by transcription factors (TF). However, the mechanisms and functions of TF mitotic bookmarking during early embryogenesis remain poorly understood. In this study, taking advantage of the naturally synchronized mitoses of Drosophila early embryos, we provide evidence that the pioneer-like transcription factor GAF acts as stable mitotic bookmarker during zygotic genome activation. We report that GAF remains associated to a large fraction of its interphase targets including at cis-regulatory sequences of key developmental genes, with both active and repressive chromatin signatures. GAF mitotic targets are globally accessible during mitosis and are bookmarked via histone acetylation (H4K8ac). By monitoring the kinetics of transcriptional activation in living embryos, we provide evidence that GAF binding establishes competence for rapid activation upon mitotic exit.

Glucocorticoid signaling induces transcriptional memory and universally reversible chromatin changes

Glucocorticoids are stress hormones that elicit cellular responses by binding to the glucocorticoid receptor, a ligand-activated transcription factor. The exposure of cells to this hormone induces wide-spread changes in the chromatin landscape and gene expression. Previous studies have suggested that some of these changes are reversible whereas others persist even when the hormone is no longer around. However, when we examined chromatin accessibility in human airway epithelial cells after hormone washout, we found that the hormone-induced changes were universally reversed after 1 d. Moreover, priming of cells by a previous exposure to hormone, in general, did not alter the transcriptional response to a subsequent encounter of the same cue except for one gene, ZBTB16, that displays transcriptional memory manifesting itself as a more robust transcriptional response upon repeated hormone stimulation. Single-cell analysis revealed that the more robust response is driven by a higher probability of primed cells to activate ZBTB16 and by a subset of cells that express the gene at levels that are higher than the induction levels observed for naïve cells.