Tissue-specific epigenetic bookmarking to preserve transcriptional programs through mitosis

My thesis addresses a fundamental problem in epigenetics and cellular memory, i.e. the mechanisms by which transcriptional information is transmitted through mitosis when transcription is transiently shut down globally. I found, by live cell confocal imaging, that the hematopoietic master transcription factor GATA1 is retained focally on chromatin during mitosis. Using a novel FACS-based approach to generate almost 100% pure mitotic cells combined with ChIP-seq I defined the mitotic occupancy pattern of GATA1 genome-wide. A notable result is that mitotic bookmarking by GATA1 favors genes encoding key hematopoietic regulatory factors but not structural genes or the globin genes. This suggests that GATA1 marks these genes to stably maintain hematopoietic transcription patterns. ^ Essential GATA1 cofactor complexes, including FOG1, SCL/TAL1, Ldb1 and LMO2 vacate chromatin during mitosis, including those sites at which GATA1 is retained. This suggests that GATA1 functions as assembly platform for co-factors at the end of mitosis, consistent with a bookmarking function. Further, GATA1 contributes to the establishment of an open chromatin structure at numerous target sites. During mitosis these sites are maintained even in the absence of GATA1, pointing at the existence of epigenetic mechanisms that propagate open chromatin domains through mitosis. ^ To investigate a potential GATA1 bookmarking function, I developed a FACS based system that enabled me to measure in hematopoietic cells the rate of post-mitotic transcription reactivation. I found that genes marked by GATA1 in mitosis reactive faster than those that are not. Several nuclear factors are known to be retained on chromatin during mitosis but their mitosis-specific function has never been examined directly. I tested for the first time the mitosis-specific function of any transcription factor by fusing GATA1 to the mitosis-specific destruction domain (MD) of a mitotic cyclin. I introduced MD-GATA1 fusion constructs into GATA1 null erythroid precursor cells and measured the onset of post-mitotic transcription. I found that genes bookmarked by GATA1 reactivated more slowly when GATA1 was degraded during mitosis whereas non-bookmarked GATA1 target genes reactivated normally. In summary, this work provides new insights into the faithful propagation of transcription patterns through the cell cycle and has important implications for lineage fidelity and cellular reprogramming.

Tissue-specific epigenetic bookmarking to preserve transcriptional programs through mitosis

My thesis addresses a fundamental problem in epigenetics and cellular memory, i.e. the mechanisms by which transcriptional information is transmitted through mitosis when transcription is transiently shut down globally. I found, by live cell confocal imaging, that the hematopoietic master transcription factor GATA1 is retained focally on chromatin during mitosis. Using a novel FACS-based approach to generate almost 100% pure mitotic cells combined with ChIP-seq I defined the mitotic occupancy pattern of GATA1 genome-wide. A notable result is that mitotic bookmarking by GATA1 favors genes encoding key hematopoietic regulatory factors but not structural genes or the globin genes. This suggests that GATA1 marks these genes to stably maintain hematopoietic transcription patterns. ^ Essential GATA1 cofactor complexes, including FOG1, SCL/TAL1, Ldb1 and LMO2 vacate chromatin during mitosis, including those sites at which GATA1 is retained. This suggests that GATA1 functions as assembly platform for co-factors at the end of mitosis, consistent with a bookmarking function. Further, GATA1 contributes to the establishment of an open chromatin structure at numerous target sites. During mitosis these sites are maintained even in the absence of GATA1, pointing at the existence of epigenetic mechanisms that propagate open chromatin domains through mitosis. ^ To investigate a potential GATA1 bookmarking function, I developed a FACS based system that enabled me to measure in hematopoietic cells the rate of post-mitotic transcription reactivation. I found that genes marked by GATA1 in mitosis reactive faster than those that are not. Several nuclear factors are known to be retained on chromatin during mitosis but their mitosis-specific function has never been examined directly. I tested for the first time the mitosis-specific function of any transcription factor by fusing GATA1 to the mitosis-specific destruction domain (MD) of a mitotic cyclin. I introduced MD-GATA1 fusion constructs into GATA1 null erythroid precursor cells and measured the onset of post-mitotic transcription. I found that genes bookmarked by GATA1 reactivated more slowly when GATA1 was degraded during mitosis whereas non-bookmarked GATA1 target genes reactivated normally. In summary, this work provides new insights into the faithful propagation of transcription patterns through the cell cycle and has important implications for lineage fidelity and cellular reprogramming.

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

<div><p>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 (<i>EGFR</i>, <i>KRAS</i>, <i>BRAF</i>, <i>NPM1</i>, <i>FLT3</i>, and <i>JAK2</i>). 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 <i>FLT3</i> 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.</p></div

Next-generation sequencing data analysis pipeline.

<p>Data analysis occurs in three sequential stages, pre-processing of NGS reads, variant calling, and variant annotation. Of note, large indels are detected by an examination of reads that failed to map to target regions of the reference genome and are recovered from a pool of rejected reads (“Trash”). SNVs, single nucleotide variants. CNVs, copy number variation.</p

Proposed Workflow for NGS and Single-Gene Assays.

<p>Three main decision points are highlighted. Specimens requiring an urgent turnaround time are routed directly for single-gene testing (possibly followed by NGS). Additionally, single-gene testing is performed on samples with less than 10% tumor or DNA inadequate for NGS (i.e., degraded or low quantity). In samples not meeting any of the above criteria, NGS is performed instead of single-gene testing. NGS results do not require confirmation by single-gene testing.</p

Two Specimens with Low-Allele Frequency <i>FLT3</i> Internal Tandem Duplications.

<p>In one specimen (A and B), a 24 bp internal tandem duplication (ITD) was seen in 7 out of 529 reads for an allele frequency of 1.3%. (A) Four of the reads containing insertions (purple bars) are shown using the Integrative Genomics Viewer. This specimen additionally harbored a <i>FLT3</i> D839G mutation in 45% of reads (B). A second specimen (C) harbored a 33 bp <i>FLT3</i> ITD in 12 out of 739 reads, for an allele frequency of 1.6%. Nine of the reads carrying an ITD are pictured.</p

Tumor Percentage of Specimens Analyzed by Targeted <i>EGFR</i> Tests.

<p>Cases were placed into 5 bins (<5%, 5–10%, 11–25%, 26–50%, >50% tumor cells in specimen).</p