Stephan Kadauke

Chromatin loops in gene regulation

The control of gene expression involves regulatory elements that can be very far from the genes they control. Several recent technological advances have allowed the direct detection of chromatin loops that juxtapose distant genomic sites in the nucleus. Here we review recent studies from various model organisms that have provided new insights into the functions of chromatin loops and the mechanisms that form them. We discuss the widespread impact of chromatin loops on gene activation, repression, genomic imprinting and the function of enhancers and insulators.

A Reconfigured Pattern of MLL Occupancy within Mitotic Chromatin Promotes Rapid Transcriptional Reactivation Following Mitotic Exit

Mixed lineage leukemia (MLL) and its metazoan Trithorax orthologs have been linked with the epigenetic maintenance of transcriptional activity. To identify mechanisms by which MLL perpetuates active transcription in dividing cells, we investigated its role during M phase of the cell cycle. Unlike other chromatin-modifying enzymes examined, we found that MLL associates with gene promoters packaged within condensed mitotic chromosomes. Genome-wide location analysis identified a globally rearranged pattern of MLL occupancy during mitosis in a manner favoring genes that were highly transcribed during interphase. Knockdown experiments revealed that MLL retention at gene promoters during mitosis accelerates transcription reactivation following mitotic exit. MLL tethers Menin, RbBP5, and ASH2L to its occupied sites during mitosis, but is dispensable for preserving histone H3K4 methylation. These findings implicate mitotic bookmarking as a component of Trithorax-based gene regulation, which may facilitate inheritance of active gene expression states during cell division.

Bromodomain protein Brd3 associates with acetylated GATA1 to promote its chromatin occupancy at erythroid target genes

Acetylation of histones triggers association with bromodomain-containing proteins that regulate diverse chromatin-related processes. Although acetylation of transcription factors has been appreciated for some time, the mechanistic consequences are less well understood. The hematopoietic transcription factor GATA1 is acetylated at conserved lysines that are required for its stable association with chromatin. We show that the BET family protein Brd3 binds via its first bromodomain (BD1) to GATA1 in an acetylation-dependent manner in vitro and in vivo. Mutation of a single residue in BD1 that is involved in acetyl-lysine binding abrogated recruitment of Brd3 by GATA1, demonstrating that acetylation of GATA1 is essential for Brd3 association with chromatin. Notably, Brd3 is recruited by GATA1 to both active and repressed target genes in a fashion seemingly independent of histone acetylation. Anti-Brd3 ChIP followed by massively parallel sequencing in GATA1-deficient erythroid precursor cells and those that are GATA1 replete revealed that GATA1 is a major determinant of Brd3 recruitment to genomic targets within chromatin. A pharmacologic compound that occupies the acetyl-lysine binding pockets of Brd3 bromodomains disrupts the Brd3-GATA1 interaction, diminishes the chromatin occupancy of both proteins, and inhibits erythroid maturation. Together these findings provide a mechanism for GATA1 acetylation and suggest that Brd3 “reads” acetyl marks on nuclear factors to promote their stable association with chromatin.

Mitotic bookmarking by transcription factors.

Mitosis is accompanied by dramatic changes in chromatin organization and nuclear architecture. Transcription halts globally and most sequence-specific transcription factors and co-factors are ejected from mitotic chromatin. How then does the cell maintain its transcriptional identity throughout the cell division cycle? It has become clear that not all traces of active transcription and gene repression are erased within mitotic chromatin. Many histone modifications are stable or only partially diminished throughout mitosis. In addition, some sequence-specific DNA binding factors have emerged that remain bound to select sites within mitotic chromatin, raising the possibility that they function to transmit regulatory information through the transcriptionally silent mitotic phase, a concept that has been termed “mitotic bookmarking.” Here we review recent approaches to studying potential bookmarking factors with regards to their mitotic partitioning, and summarize emerging ideas concerning the in vivo functions of mitotically bound nuclear factors.

Functions of BET proteins in erythroid gene expression

Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.

Activated protein C resistance testing for factor V Leiden

Activated protein C resistance assays can detect factor V Leiden with high accuracy, depending on the method used. Factor Xa inhibitors such as rivaroxaban and direct thrombin inhibitors including dabigatran, argatroban, and bivalirudin can cause falsely normal results. Lupus anticoagulants can cause incorrect results in most current assays. Assays that include dilution into factor V‐deficient plasma are needed to avoid interference from factor deficiencies or elevations, which can arise from a wide variety of conditions such as warfarin, liver dysfunction, or pregnancy. The pros and cons of the currently available assays are discussed. Am. J. Hematol. 89:1147–1150, 2014.

“Remembering” tissue-specific transcription patterns through mitosis

During cell growth, tissue-specific gene expression programs are maintained through multiple rounds of cell division. Experiments performed in the 1960s showed that RNA synthesis stops during late prophase and restarts in telophase of mitosis,1 raising the fundamental question as to how transcriptional information is preserved through the mitotic phase of the cell cycle. While the exact mechanisms that lead to mitotic repression of transcription are still under debate, they are known to involve pervasive phosphorylation of chromatin, chromosome condensation and premature termination of transcription.2 Simultaneously, there is a widespread displacement of the basal transcription machinery, gene-specific transcription factors and co-factors, chromatin remodelers and modifying enzymes as well as factors that recognize and bind to specific chromatin modifications. However, evidence is emerging that select nuclear factors and histone modifications are retained on mitotic chromatin.3,4 It has long been hypothesized that mitotic retention of nuclear factors may function to mark genes in a way that enables reassembly of transcription complexes after mitosis. This proposed mitotic memory mechanism has been dubbed “bookmarking.” A small number of mitotically retained factors, including MLL and BRD4, have been shown to function as molecular bookmarks by facilitating post-mitotic transcription re-initiation of their mitotic target genes.5-7 The hematopoietic zinc finger transcription factor GATA1 controls the expression of virtually all erythroid-specific genes8 and is critical for establishing and maintaining the erythroid compartment. In our recent study9 we report that, using live-cell imaging, a small fraction of GATA1 is retained on chromatin during mitosis. We next aimed to define the genome-wide occupancy pattern of GATA1 during mitosis using ChIP-seq. To obtain highly purified mitotic cell populations for ChIP-seq analysis, we developed a novel FACS-based approach that exploits the widespread serine 10 phosphorylation of histone H3 during mitosis. The results revealed that GATA1 is preferentially retained at a subset of genes encoding key hematopoietic nuclear regulatory factors, suggesting that GATA1 bookmarking contributes to the maintenance of hematopoietic transcription patterns. This idea is further supported by our finding that genes marked by GATA1 in mitosis tend to reactivate faster than those that are not. To test directly whether GATA1 performs a mitosis-specific function on these genes, we established a system in which GATA1 levels are nearly normal in interphase, but selectively deficient in mitosis. To this end, we generated a version of GATA1 that is destroyed in mitosis by fusing it to the mitotic destruction domain (MD) of cyclin B1. MD-GATA1 fusion constructs were introduced into GATA1-null erythroid precursor cells, which are dependent upon exogenous GATA1 for differentiation. We then measured the kinetics of post-mitotic transcription reactivation of GATA1 target genes. Genes bookmarked by GATA1 reactivated more slowly when GATA1 was degraded during mitosis, whereas non-bookmarked GATA1 target genes reactivated normally. Additionally, mitotic destruction of GATA1 also led to partial de-repression of bookmarked genes that are normally inhibited by GATA1. To our knowledge, this represents the first direct demonstration of a mitosis-specific function for any transcription factor. This approach should be superior to conventional knockout or knockdown experiments since results from the latter might be confounded by effects outside of mitosis. Like most nuclear factors, GATA1 relies on co-factors for its ability to bind to target sites and regulate transcriptional activity. Notably, none of the examined tissue-specific GATA1 co-factors (FOG1, SCL/TAL1, Ldb1 and LMO2) were found on mitotic chromosomes, regardless of whether GATA1 was retained at these sites. However, other GATA1 co-factors might regulate GATA1 binding to mitotic chromatin. One particularly interesting candidate is the widely expressed protein Brd3, which associates with acetylated GATA1.10 Like the closely related mitotic bookmarking factor Brd4, strong mitotic retention was observed with Brd3 (unpublished observations). Future work will examine whether Brd3 plays a role in mitotic GATA1 bookmarking. Important questions that remain to be addressed include: (1) What distinguishes sites that are bound by GATA1 in mitosis from those that are not? (2) Do sequences that retain GATA1 during mitosis function autonomously, i.e., when integrated at heterologous genomic sites? (3) If so, do they convey rapid reactivation on a linked reporter gene, and can this approach be used to pinpoint critical DNA sequence elements and/or chromatin features that can facilitate or repress mitotic GATA1 retention? While preliminary studies have not yet identified features that reliably discriminate between mitotically occupied vs. vacated sites, certain trends became apparent. For example, clustering of GATA1 binding motifs, H3K4 trimethylation and promoter-proximal location occur more frequently near mitotically maintained GATA1 binding sites. A more general question is whether rapid post-mitotic reactivation of genes, as well as the repression of lineage- or differentiation stage-inappropriate genes, is important for lineage stability. In other words, could failure to bookmark genes facilitate lineage reprogramming? We speculate that synchronizing transcriptional reactivation after mitosis and maintaining gene repression might be a general mechanism to suppress cell-to-cell variability in gene expression, thus stabilizing cell identity. Pulsing erythroid cells that contain wild-type or mitotically unstable GATA1 with lineage reprogramming factors might be a way to test this idea directly. In summary, this work provides new insights into the faithful propagation of transcription patterns through the cell cycle with potential implications for lineage fidelity and cellular reprogramming. We hope that the versatile systems we have established for this study, such as the FACS-based purification of mitotic cells as well as the mitosis-specific destruction of a nuclear factor, will aid future investigation into mechanisms of mitotic bookmarking.

Building a Robust Tumor Profiling Program: Synergy between Next-Generation Sequencing and Targeted Single-Gene Testing

Next-generation sequencing (NGS) is a powerful platform for identifying cancer mutations. Routine clinical adoption of NGS requires optimized quality control metrics to ensure accurate results. To assess the robustness of our clinical NGS pipeline, we analyzed the results of 304 solid tumor and hematologic malignancy specimens tested simultaneously by NGS and one or more targeted single-gene tests (EGFR, KRAS, BRAF, NPM1, FLT3, and JAK2). For samples that passed our validated tumor percentage and DNA quality and quantity thresholds, there was perfect concordance between NGS and targeted single-gene tests with the exception of two FLT3 internal tandem duplications that fell below the stringent pre-established reporting threshold but were readily detected by manual inspection. In addition, NGS identified clinically significant mutations not covered by single-gene tests. These findings confirm NGS as a reliable platform for routine clinical use when appropriate quality control metrics, such as tumor percentage and DNA quality cutoffs, are in place. Based on our findings, we suggest a simple workflow that should facilitate adoption of clinical oncologic NGS services at other institutions.

Case 36-2017

A 30-year-old man presented with fatigue, rash, anemia, and thrombocytopenia. Three years earlier, after an automobile accident, abdominal-wall hematomas, anemia, thrombocytopenia, and hematuria had developed but spontaneously resolved. A diagnostic test was performed.

Utilization Management of Blood Derivatives

The blood bank is unique from a laboratory utilization management perspective, as it dispenses therapeutic products which not only incur costs but also pose direct risks to patients. Like traditional blood components such as red blood cells and platelets, blood “derivatives” including intravenous immunoglobulin, albumin, and clotting factor concentrates tend to be overutilized. In contrast to regular blood components, derivatives tend to be dispensed in low volumes, carry a high per-order cost, and have a complex structure of indications. Gatekeeping interventions, if implemented in collaboration with the leadership of the affected clinical departments, can be effective for managing utilization of blood derivatives. Examples of successful interventions targeting blood derivative use are discussed.