Spt6 promotes transcription elongation at many genes and functions as a histone H3 chaperone to alter chromatin structure during transcription. We show here that mammalian Spt6 binds Ser2-phosphorylated (Ser2P) RNA polymerase II (RNAPII) through a primitive SH2 domain, which recognizes phosphoserine rather than phosphotyrosine residues. Surprisingly, a point mutation in the Spt6 SH2 domain (R1358K) blocked binding to RNAPIIo without affecting transcription elongation rates in vitro. However, HIV-1 and c-myc RNAs formed in cells expressing the mutant Spt6 protein were longer than normal and contained splicing defects. Ectopic expression of the wild-type, but not mutant, Spt6 SH2 domain, caused bulk poly(A) + RNAs to be retained in the nucleus, further suggesting a widespread role for Spt6 in mRNA processing or assembly of export-competent mRNP particles. We cloned the human Spt6-interacting protein, hIws1 (interacts with Spt6), and found that it associates with the nuclear RNA export factor, REF1/Aly. Depletion of endogenous hIws1 resulted in mRNA processing defects, lower levels of REF1/Aly at the c-myc gene, and nuclear retention of bulk HeLa poly(A) + RNAs in vivo. Thus binding of Spt6 to Ser2-P RNAPII provides a cotranscriptional mechanism to recruit Iws1, REF1/Aly, and associated mRNA processing, surveillance, and export factors to responsive genes.[Keywords: Spt6; SH2 domain; RNAPII CTD; transcription elongation; splicing; nuclear mRNA export] Supplemental material is available at http://www.genesdev.org.

HIV-1 Tat binds human CyclinT1 and recruits the CDK9/P-TEFb complex to the viral TAR RNA in a step that links RNA polymerase II (RNAPII) C-terminal domain (CTD) Ser 2 phosphorylation with transcription elongation. Previous studies have suggested a connection between Tat and pre-mRNA splicing factors. Here we show that the splicing-associated c-Ski-interacting protein, SKIP, is required for Tat transactivation in vivo and stimulates HIV-1 transcription elongation, but not initiation, in vitro. SKIP associates with CycT1:CDK9/P-TEFb and Tat:P-TEFb complexes in nuclear extracts and interacts with recombinant Tat:P-TEFb:TAR RNA complexes in vitro, indicating that it may act through nascent RNA to overcome pausing by RNAPII. SKIP also associates with U5snRNP proteins and tri-snRNP110K in nuclear extracts, and facilitates recognition of an alternative Tat-specific splice site in vivo. The effects of SKIP on transcription elongation, binding to P-TEFb, and splicing are mediated through the SNW domain. HIV-1 Tat transactivation is accompanied by the recruitment of P-TEFb, SKIP, and tri-snRNP110K to the integrated HIV-1 promoter in vivo, whereas the U5snRNPs associate only with the transcribed coding region. These findings suggest that SKIP plays independent roles in transcription elongation and pre-mRNA splicing.[Keywords: HIV-1 Tat; CycT1:CDK9/P-TEFb; c-Ski-interacting protein; transcription elongation; alternative splicing; HIV-1 TAR RNA] Supplemental material is available at http://www.genesdev.org.

SUMMARY The Ski-interacting protein, SKIP/SNW1, associates with the P-TEFb/CDK9 elongation factor and coactivates inducible genes, including HIV-1. We show here that SKIP also associates with c-Myc and Menin, a subunit of the MLL1 histone methyltransferase (H3K4me3) complex, and that HIV-1 Tat transactivation requires c-Myc and Menin, but not MLL1 or H3K4me3. RNAi-ChIP experiments reveal that SKIP acts downstream of Tat:P-TEFb to recruit c-Myc and its partner TRRAP, a scaffold for histone acetyltransferases, to the HIV-1 promoter. By contrast, SKIP is recruited by the RNF20 H2B ubiquitin ligase to the basal HIV-1 promoter, in a step that is bypassed by Tat and down-regulated by c-Myc. Interestingly, we find that SKIP and P-TEFb are dispensable for UV stress-induced HIV-1 transcription, which is strongly up-regulated by treating cells with the CDK9 inhibitor, flavopiridol. Thus SKIP acts with c-Myc and Menin to promote HIV-1 Tat:P-TEFb transcription at an elongation step that is bypassed under stress.

Changes in DNA methylation, causing chromosome instability and altered gene expression, have been strongly associated with carcinogenesis. Due to the involvement of methylation in cancer, methylation profiles have been heralded as promising cancer biomarkers. Here, we present a primer design pipeline and an analysis workflow that we have developed to design and analyze custom methylation panels and detect methylation status. An automated primer design pipeline for methylation sequencing has been developed, consisting of genome conversion, primer selection, amplicon tiling, and generation of optimal amplicons. Custom methylation panels can be designed using pre-converted genomes or reference genome sequences for any other organism which can then be converted. The pipeline has the capability to create custom targeted panels specific to any methylation sites of interest. The pipeline designs Ion AmpliSeq primers to enable high multiplexing and robust amplification of low abundance or degraded DNA. Following the creation of a custom panel, a complete 3-day workflow has been developed, comprising bisulfite conversion, library construction, template preparation, sequencing and data analysis. This 3-day protocol offers manual or automated library options, low input (10-20ng DNA) and a flexible multiplexed approach with quantitative information at single base pair resolution. Sequencing is performed on the Ion GeneStudio S5 system. The bioinformatics analysis has been streamlined into a downloadable plugin performing alignment and DNA methylation calling for amplicons on both the Watson and Crick strands. To evaluate the in silico performance of the primer design pipeline for targeted bisulfite sequencing, a custom methylation panel was created using a set of 48 oncology markers from the BLUEPRINT consortium. These markers were also used for the Ion AmpliSeq Methylation Panel for Cancer Research, which was compared to the custom methylation panel to evaluate the performance of the pipeline. Key metrics from in silico design such as total number of degenerate oligos, mean amplicon length and average Tm spread for the custom methylation panel are equal to or better than Ion AmpliSeq Methylation Panel for Cancer Research. To assess the sequencing performance of the panel, two control gDNA samples were used. The expected average methylation status across all CpGs were >98% and <5% for the first sample and the second sample, respectively. The evaluation was also carried out with an equal mixture of these two samples. The wet lab testing of the custom methylation panel generated comparable results to the Ion AmpliSeq Methylation Panel for Cancer Research. The primer design pipeline and 3-day workflow provide custom design of targeted methylation panels along with quantitative analysis of relevant oncology markers from low DNA input. Citation Format: Zunping Luo, Loni Pickle, Andrew Hatch, Aren Ewing, Fiona Hyland, David Berman, Palak Patel, Mark Andersen. Custom primer design pipeline and analysis workflow for targeted methylation sequencing using NGS Ion AmpliSeq technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 158.

Background B cell repertoire analysis by next-generation sequencing (NGS) has shown particular utility in the field of hematological oncology research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to flow-based methods, and the elimination of specifically designed primers often required for qPCR-based methods. Owing to primer-primer interactions and incompatibility of reaction conditions, current multiplex PCR assays require separate PCR reactions to survey each immunoglobulin chain (IGH, IGK, IGL), often leading to a longer time-to-answer for samples in which no marker is initially detected. We have developed an assay for receptor analysis based on Ion AmpliSeq technology to circumvent these issues, allowing the effective use of up to thousands of primers in a single reaction. The highly multiplexed, pan-clonality NGS assay provides for efficient detection of IGH, IGK, and IGL chain rearrangements in a single reaction. Methods We developed a single primer panel targeting the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IGK, IGL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IGK loci rearrangements involving Kappa deletion and C intron elements. To evaluate performance, we conducted clonality assessment and limit-of-detection testing used gDNA from a total of 45 research samples representing common B cell malignancies. We included samples derived from peripheral blood, bone marrow, and FFPE-preserved tissues at input levels ranging from 100ng to 2µg. Finally, we further characterized the samples via a separate AmpliSeq-based multiplex PCR assay targeting rearranged TCRB and TCRG chains. Sequencing and clonality analysis was performed using the Ion GeneStudio S5 System and Ion Reporter 5.16. Results Clonality assessments carried out using gDNA collected from both cell line and clinical research samples (CLL, B-ALL, Multiple Myeloma, Burkitt's Lymphoma, NHL, and DLBCL) show a >90% overall positive detection rate. Assessment of linearity-of-response and limit-of-detection was carried out using cell lines diluted in PBL to between 10-3 and 10-6 by mass. The multi-receptor assay performs as expected, with linear response to the cell line frequency across the range tested, including the ability to detect clones of interest at 10-6. Conclusions These results demonstrate the robustness of our newly developed Ion AmpliSeq-assay for B cell receptor heavy and light chains. We expect this assay to simplify the workflow for clonality assessment and rare clone detection in B cell malignancy research. For research use only. Disclosures No relevant conflicts of interest to declare.

Induced pluripotent stem cells (iPSCs) hold immense potential in disease modeling, drug discovery and regenerative medicine. Despite advances in reprogramming methods, generation of clinical-grade iPSCs remains a challenge. Reported here is the first off-the-shelf reprogramming kit, CTS CytoTune-iPS 2.1, specifically designed for clinical and translational research. Workflow gaps were identified, and methods developed were used to consistently generate iPSC from multiple cell types. Resulting clones were subjected to characterization that included confirmation of pluripotency, preservation of genomic integrity and authentication of cell banks via an array of molecular methods including high resolution microarray and next-generation sequencing. Development of integrated xeno-free workflows combined with comprehensive characterization offers generation of high-quality iPSCs that are suited for clinical and translational research.

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Introduction: B cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. Methods: A B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. Primers are included to amplify rearrangements involving Kappa deletion element. Reproducibility studies and clonality assessment were conducted using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal via flow cytometry or IHC/ISH in tissue sections. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software. Results: Clonality assessment of MM research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples contained somatic hypermutation with 6 of 45 samples showing mutation rates greater than 10%. Lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Conclusions: These results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify workflow and inclusion of analysis tools such as automated somatic hypermutation rate determination and clonal lineage identification to open new paths for research in B cell disorders. Citation Format: Geoffrey Marc Lowman, Landon Pastushok, Karen Mochoruk, Wayne Hill, Michelle Toro, Loni Pickle, Carolina Gonzalez, Stephanie Ostresh, Shrutii Sarda, Chenchen Yang, Julie Stakiw, Mark Bosch, Hadi Goubran, Ron Geyer, John DeCoteau. Evaluation of multiple myeloma research samples by analysis of B cell heavy and light chain receptors in a single NGS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2293.