Context-Dependent Gene Regulation by Homeodomain Transcription Factor Complexes Revealed by Shape-Readout Deficient Proteins.

Eukaryotic transcription factors (TFs) form complexes with various partner proteins to recognize their genomic target sites. Yet, how the DNA sequence determines which TF complex forms at any given site is poorly understood. Here, we demonstrate that high-throughput in vitro DNA binding assays coupled with unbiased computational analysis provide unprecedented insight into how different DNA sequences select distinct compositions and configurations of homeodomain TF complexes. Using inferred knowledge about minor groove width readout, we design targeted protein mutations that destabilize homeodomain binding both in vitro and in vivo in a complex-specific manner. By performing parallel systematic evolution of ligands by exponential enrichment sequencing (SELEX-seq), chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing (RNA-seq), and Hi-C assays, we not only classify the majority of in vivo binding events in terms of complex composition but also infer complex-specific functions by perturbing the gene regulatory network controlled by a single complex.

Can aggressive cancers be identified by the "aggressiveness" of their chromatin?

Phenotypic plasticity is a crucial feature of aggressive cancer, providing the means for cancer progression. Stochastic changes in tumor cell transcriptional programs increase the chances of survival under any condition. I hypothesize that unstable chromatin permits stochastic transitions between transcriptional programs in aggressive cancers and supports non-genetic heterogeneity of tumor cells as a basis for their adaptability. I present a mechanistic model for unstable chromatin which includes destabilized nucleosomes, mobile chromatin fibers and random enhancer-promoter contacts, resulting in stochastic transcription. I suggest potential markers for "unsettled" chromatin in tumors associated with poor prognosis. Although many of the characteristics of unstable chromatin have been described, they were mostly used to explain changes in the transcription of individual genes. I discuss approaches to evaluate the role of unstable chromatin in non-genetic tumor cell heterogeneity and suggest using the degree of chromatin instability and transcriptional noise in tumor cells to predict cancer aggressiveness.

Investigating cell cycle-dependent gene expression in the context of nuclear architecture at single-allele resolution.

Nuclear architecture is the organization of the genome within a cell nucleus with respect to different nuclear landmarks such as the nuclear lamina, nuclear matrix or nucleoli. Recently, nuclear architecture has emerged as a major regulator of gene expression in mammalian cells. However, studies connecting nuclear architecture with gene expression are largely population-averaged and do not report on the heterogeneity in genome organization or gene expression within a population. In this report we present a method for combining 3D DNA fluorescence in situ hybridization (FISH) with single-molecule RNA FISH (smFISH) and immunofluorescence to study nuclear architecture-dependent gene regulation on a cell-by-cell basis. We further combine our method with imaging-based cell cycle staging to correlate nuclear architecture with gene expression across the cell cycle. We present this in the context of the cyclin-A2 (CCNA2) gene, which has known cell cycle-dependent expression. We show that, across the cell cycle, the expression of a CCNA2 gene copy is stochastic and depends neither on its sub-nuclear position - which usually lies close to nuclear lamina - nor on the expression from other copies of the gene.This article has an associated First Person interview with the first author of the paper.

Fluorescent labeling of genomic loci in Drosophila imaginal discs with heterologous DNA-binding proteins.

Using the Drosophila melanogaster Hox gene Ultrabithorax (Ubx) as an example, we demonstrate the use of three heterologous DNA-binding protein systems-LacI/LacO, ParB1/ParS1, and ParB2/ParS2-to label genomic loci in imaginal discs with the insertion of a small DNA tag. We compare each system, considering the impact of labeling in genomic regions (1) inside versus outside of a transcribed gene body and (2) with varying chromatin accessibility. We demonstrate the value of this system by interrogating the relationship between gene expression level and enhancer-promoter distance, as well as inter-allelic distance at the Ubx locus. We find that the distance between an essential intronic cis-regulatory element, anterobithorax (abx), and the promoter does not vary with expression level. In contrast, inter-allelic distance correlates with Ubx expression level.

Parent-of-origin effects on nuclear chromatin organization and behavior in a Drosophila model for Williams-Beuren Syndrome.

Prognosis of neuropsychiatric disorders in progeny requires consideration of individual (1) parent-of-origin effects (POEs) relying on (2) the nerve cell nuclear 3D chromatin architecture and (3) impact of parent-specific miRNAs. Additionally, the shaping of cognitive phenotypes in parents depends on both learning acquisition and forgetting, or memory erasure. These processes are independent and controlled by different signal cascades: the first is cAMPdependent, the second relies on actin remodeling by small GTPase Rac1 - LIMK1 (LIM-kinase 1). Simple experimental model systems such as Drosophila help probe the causes and consequences leading to human neurocognitive pathologies. Recently, we have developed a Drosophila model for Williams-Beuren Syndrome (WBS): a mutant agnts3 of the agnostic locus (X:11AB) harboring the dlimk1 gene. The agnts3 mutation drastically increases the frequency of ectopic contacts (FEC) in specific regions of intercalary heterochromatin, suppresses learning/memory and affects locomotion. As is shown in this study, the polytene X chromosome bands in reciprocal hybrids between agnts3 and the wild type strain Berlin are heterogeneous in modes of FEC regulation depending either on maternal or paternal gene origin. Bioinformatic analysis reveals that FEC between X:11AB and the other X chromosome bands correlates with the occurrence of short (~30 bp) identical DNA fragments partly homologous to Drosophila 372-bp satellite DNA repeat. Although learning acquisition in a conditioned courtship suppression paradigm is similar in hybrids, the middle-term memory formation shows patroclinic inheritance. Seemingly, this depends on changes in miR-974 expression. Several parameters of locomotion demonstrate heterosis. Our data indicate that the agnts3 locus is capable of trans-regulating gene activity via POEs on the chromatin nuclear organization, thereby affecting behavior.