Paul Knoepfler

University of California, Davis | UCD · Department of Cell Biology and Human Anatomy

The histone variant H3.3 K27M mutation is common in diffuse midline gliomas (DMG) and is associated with poor prognosis. While some insights into K27M epigenetic functions have emerged, much less is known about how K27M impacts chromatin structure and function, and the resulting transcriptomic consequences. Recently, we developed isogenic CRISPR-ed...

Histone variant H3.3 plays novel roles in development as compared to canonical H3 proteins and is the most commonly mutated histone protein of any kind in human disease. Here we discuss how gene targeting studies of the two H3.3-coding genes H3f3a and H3f3b have provided important insights into H3.3 functions including in gametes as well as brain a...

Histone variant H3.3 is encoded by two genes, H3f3a and H3f3b, which can be expressed differentially depending on tissue type. Previous work in our lab has shown that knockout of H3f3b causes some neonatal lethality and infertility in mice, and chromosomal defects in mouse embryonic fibroblasts (MEFs). Studies of H3f3a and H3f3b null mice by others...

Background The histone variant H3.3 K27M mutation is a defining characteristic of diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). This histone mutation is responsible for major alterations to histone H3 post-translational modification (PTMs) and subsequent aberrant gene expression. However, much less is known about the effect...

Many gene networks are shared between pluripotent stem cells and cancer; a concept exemplified by several DPPA factors such as DPPA2 and DPPA4, which are highly and selectively expressed in stem cells but also found to be reactivated in cancer. Despite their striking expression pattern, for many years the function of DPPA2 and DPPA4 remained a myst...

Aim: There is a critical need for safe and effective treatments for COVID-19. One possible type of treatment is cellular medicine such as stem cell therapy, but its potential is unclear. Here, our aim was to assess the potential impact of the many cellular medicine trials for COVID-19. Materials & methods: We collected and analyzed data for defined...

Histone H3.3 mutations are a hallmark of pediatric gliomas, but their core oncogenic mechanisms are not well-defined. To identify major effectors, we used CRISPR-Cas9 to introduce H3.3K27M and G34R mutations into previously H3.3-wildtype brain cells, while in parallel reverting the mutations in glioma cells back to wildtype. ChIP-seq analysis broad...

Alterations in histone H3.3 are common driver mutations in high-grade pediatric gliomas, but the central oncogenic mechanisms remain an open question. To identify important mutant H3.3 effectors, we used CRISPR-Cas9 to precisely introduce H3.3 K27M and G34R mutations into previously H3.3-wildtype human astrocyte and glioma cells, while in parallel...

Aim: The industry of unproven stem cell clinics has rapidly mushroomed throughout the USA, posing risks to patients and the research field. In this study, the aim was to better define how this industry changes. Methods: I analyzed a large cohort of US stem cell clinic firms and their distinct clinic locations as defined in 2015–2016 for their statu...

Pediatric high-grade gliomas often contain mutations in the H3F3A gene encoding the histone variant H3.3 and more rarely in canonical histone H3 family genes, a feature distinguishing them from adult gliomas. To define specific functionally significant changes in epigenomic states driven by mutant H3.3, we have utilized CRISPR-Cas9 to introduce spe...

The stem cell and regenerative medicine arena has become increasingly complicated in recent years with thousands of people involved. There are as many as a dozen or more main groups of stakeholders, who together may be viewed as one ecosystem that is now rapidly evolving. The nature of the ecosystem and its evolution have major implications for not...

Developmental pluripotency associated factor 4 (Dppa4) is a highly specific marker of pluripotent cells, and is also overexpressed in certain cancers, but its function in either of these contexts is poorly understood. In this study, we use ChIP-Seq to identify Dppa4 binding genome-wide in three distinct cell types: mouse embryonic stem cells (mESC)...

Regulators are now more often distinguishing between perceived good citizens and “bad actors” in stem cell and regenerative medicine clinical research, resulting in relatively more polar, carrot-and-stick oversight approaches. Here, I discuss why there may be too much carrot and not enough stick by regulators for effective enforcement. Regulators a...

Wie würde man vorgehen, um einen GMO sapiens zu erschaffen? Wäre es schwierig? Welche Schritte wären dafür erforderlich? Wie viele Vorkenntnisse müsste man haben? Könnte man es als Do-it-yourself-Projekt betrachten, vielleicht sogar in der eigenen Garage, mit einer bei eBay erstandenen Ausrüstung?

Developmental Pluripotency-Associated-4 (DPPA4) is one of the few core pluripotency genes lacking clearly defined molecular and cellular functions. Here we used a proteomics screening approach of human embryonic stem cell (hESC) nuclear extract to determine DPPA4 molecular functions through identification of novel cofactors. Unexpectedly, the signa...

Hundreds of businesses in the US currently advertise a wide range of non-US FDA-approved stem cell interventions. Here we present a novel systematic temporal analysis of US companies engaged in direct-to-consumer marketing of putative stem cell treatments. Between 2009 and 2014, the number of new US stem cell businesses with websites grew rapidly,...

The transcriptional functions of the class I histone deacetylases (HDACs) HDAC1 and HDAC2 are mainly viewed as both repressive and redundant based on murine knockout studies, but they may have additional independent roles and their physiological functions in human cells are not as clearly defined. To address the individual epigenomic functions of H...

HDAC2 disruption reduces HDAC3, but not HDAC1: Individual clone data. (A) Quantitation of individual clones shown in Fig 1B. HDAC2 protein levels normalized to B-actin were quantified through the LiCOR imaging software and plotted relative to WT. (B) Nuclear lysates of a panel of 9 additional HDAC2-targeted clonal lines were analyzed for HDAC2 prot...

Predicted off-target regions do not have Indels. Genomic DNA was isolated from WT cells or HDAC2 null clones #5, #14, and #15 and PCR-amplified for the top four predicted off-target regions based on sequence complementarity to the gRNA sequence, as predicted by the website tool. The predicted off-target regions in intronic regions of BMP15 (A), PAR...

PCA of WT and clonal lines. The DESeq2 R package was used to perform and plot PCA. (TIF)

HDAC2 null lines exhibit increased H4ac enrichment at a few target genes. ChIP-qPCR was conducted on crosslinked chromatin of WT and clones #5, #14, and #15 with antibodies to pan-H4ac or IgG control and SYBR green qPCR analysis was conducted for HDAC2-repressed (A) or HDAC2-activated (B) gene targets to evaluate the binding of HDAC2 or HDAC1 or en...

Tested off-target sites as predicted by crispr.mit.edu. (DOCX)

Loss of HDAC2 does not alter global H3K9 or H4 modifications. (A-C) Histone acid extracts of WT or HDAC2 null clones #5, #14, and #15 were analyzed for total H4ac (A), H3K9ac (B), or H3K9me3 (C) levels through Western blotting. Image is representative of three independent Western blots. (D-F) Quantitation of the triplicate Western blot results: H4a...

qPCR assay primers. (DOCX)

Expression patterns of HDAC2 target genes are not consistently altered in the same ways simply by clonal selection and puro exposure absent HDAC2 disruption as compared to RNA-Seq defined expression changes in HDAC2 knockouts. RNA isolated from 293FT cells transfected with empty pCas9-puro and clonally selected with puromycin was subjected to qPCR...

Off-target region PCR primers. (DOCX)

Quality of RNA-Seq reads as determined through RSeQC. Duplication rates (Dup) were determined both by Position (Pos) and Sequence (Seq). (DOCX)

ChIP-qPCR assay primers. (DOCX)

ENCODE HDAC2 ChIP-seq peaks in Tier 1 cell lines present at validated HDAC2 targets. (DOCX)

Differentially expressed genes in HDAC CRISPR lines. (XLSX)

GO analysis of HDAC2 upregulated and downregulated genes. (XLS)

Human pluripotent stem cells (hPSC) have great clinical potential via the use of their differentiated progeny, a population in which there is some concern over risks of tumorigenicity or other unwanted cellular behavior due to residual hPSC. Preclinical studies using human stem cells are most often performed within a xenotransplant context. Here, w...

The goal of editing the genomes of stem cells to generate model organisms and cell lines for genetic and biological studies has been pursued for decades. There is also exciting potential for future clinical impact in humans. While recent, rapid advances in targeted nuclease technologies have led to unprecedented accessibility and ease of gene editi...

Significance: This article focuses on the strategies used to recruit patients via for-profit stem cell clinic seminars and the marketing claims made as well as the impact on the public and the stem cell translational medicine field. Although the field is very aware of the existence of such seminars, this piece is significant and novel for reportin...

Direct-to-consumer marketing of unapproved stem cell interventions is a well-known phenomenon in countries with lax medical regulations. However, an examination of Internet-based marketing claims revealed widespread promotion of such interventions by businesses based in the United States. Such commercial activity suggests that regulatory agencies m...

Post-publication peer review (PPPR) is transforming how the life sciences community evaluates published manuscripts and data. Unsurprisingly, however, PPPR is experiencing growing pains, and some elements of the process distinct from standard pre-publication review remain controversial. I discuss the rapid evolution of PPPR, its impact, and the cha...

The phrase "bench to bedside" is commonly used to describe the translation of basic discoveries such as those on stem cells to the clinic for therapeutic use in human patients. However, there is a key intermediate step in between the bench and the bedside involving governmental regulatory oversight such as by the Food and Drug Administration (FDA)...

The histone variant H3.3 is involved in diverse biological processes, including development, transcriptional memory and transcriptional reprogramming, as well as diseases, including most notably malignant brain tumors. Recently, we developed a knockout mouse model for the H3f3b gene, one of two genes encoding H3.3. Here, we show that targeted disru...

The transcription factor Miz-1 can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein. The genomic binding of Miz-1 includes both core promoters and more distal sites, but the preferred DNA binding motif of Miz-1 has been unclear. We used a high-throughput in vitro technique, Bind-n-Seq, to ide...

One of the biggest roadblocks to using stem cells as the basis for regenerative medicine therapies is the tumorigenicity of stem cells. Unfortunately, the unique abilities of stem cells to self-renew and differentiate into a variety of cell types are also mechanistically linked to their tumorigenic behaviors. Understanding the mechanisms underlying...

A host of cancer types exhibit aberrant histone modifications. Recently, distinct and recurrent mutations in a specific histone variant, histone H3.3, have been implicated in a high proportion of malignant pediatric brain cancers. The presence of mutant H3.3 histone disrupts epigenetic posttranslational modifications near genes involved in cancer p...

In order to identify novel pluripotency-related oncogenes, an expression screen for oncogenic foci-inducing genes within a retroviral human embryonic stem cell (hESC) cDNA library was conducted. From this screen, we identified not only known oncogenes but also intriguingly the key pluripotency factor, DPPA4 (developmental pluripotency-associated 4)...

Abstract The stem cell field is at a critical juncture in late 2013. We find ourselves buoyed by building momentum for both transformative basic science discoveries and clinical translation of stem cells. Cellular reprogramming has given the field exciting new avenues as well. The overall prospect of novel stem cell-based therapies becoming a reali...

There is no rulebook for how to be a scientist advocate, but that shouldn't stop researchers from engaging with the broader community. In fact, it's an essential part of being a good steward of your science.

Myc and N-Myc have widespread impacts on the chromatin state within cells, both in a gene-specific and genome-wide manner. Our laboratory uses functional genomic methods including chromatin immunoprecipitation (ChIP), ChIP-chip, and, more recently, ChIP-seq to analyze the binding and genomic location of Myc. In this chapter, we describe an effectiv...

In order to elucidate the function of Myc in the maintenance of pluripotency and self-renewal in mouse embryonic stem cells (mESCs), we screened for novel embryonic stem cell (ESC)-specific interactors of Myc by mass spectrometry. Undifferentiated embryonic cell transcription factor 1 (Utf1) was identified in the screen as a putative Myc binding pr...

Background The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encod...

Stem cell-based regenerative and cellular medicine is an exciting, emerging area of medical practice. While bone marrow transplantation, a stem cell-based therapy, has been a part of medicine for decades, in recent years newer and more diverse forms of stem cell-based therapies are being used to treat a rapidly growing population of patients in the...

Induced pluripotent stem cells (iPSCs) have the potential for creating patient-specific regenerative medicine therapies, but the links between pluripotency and tumorigenicity raise important safety concerns. More specifically, the methods employed for the production of iPSCs and oncogenic foci (OF), a form of in vitro produced tumor cells, are surp...

Recently in Cell, Jia et al. (2012) reported novel Utf1-controlled mechanisms of maintaining pluripotency and self-renewal in embryonic stem cells (ESCs). Utf1 buffers bivalent gene expression by competitive binding with polycomb repressive complex 2 and initiation of mRNA degradation.

Induced pluripotent stem cells are different from embryonic stem cells as shown by epigenetic and genomics analyses. Depending on cell types and culture conditions, such genetic alterations can lead to different metabolic phenotypes which may impact replication rates, membrane properties and cell differentiation. We here applied a comprehensive met...

Method comparison for quantification of select compounds in m15 progenitor cells, induced pluripotent stem cells and embryonic stem cells, detected in more than one metabolomics platform. Upper panel: unsaturated phosphatidyl-lipids detected by HILIC-QTOF MS and by nanoelectrospray-linear ion trap mass spectrometry. All data are given as normalized...

Identified lipids by shotgun lipidomics using direct infusion nanoelectrospray/linear ion trap tandem mass spectrometry and LipidBlast annotations with manual quality checks. (PDF)

Identified metabolites in m15 and stem cell cultures using gas chromatography-time of flight mass spectrometry and BinBase annotations, and liquid chromatography-quadrupole time of flight mass spectrometry and Metlin and LipidBlast annotations. Significance levels are given as one-way ANOVA p-values. (PDF)

Principal Component Analysis (PCA) and Partial Least Square (PLS) multivariate analysis on all three metabolomic platforms combined (left panels) or excluding the nanoelectrospray-ion trap MS data (right panels). (PDF)

Extended information on Materials and Methods : Cell Culture and Preparation; NanoESI-MS Extraction and Analysis; GC-TOF MS Extraction and Analysis; LC-QTOF MS Extraction and Analysis; MetaMapp Network Visualization. (PDF)

The production of human induced pluripotent stem cells (hiPSCs) has greatly expanded the realm of possible stem cell-based regenerative medicine therapies and has particularly exciting potential for autologous therapies. However, future therapies based on hiPSCs will first have to address not only similar regulatory issues as those facing human emb...

Here we examine the functions of the Myc cofactor and histone acetyltransferase, GCN5/KAT2A, in neural stem and precursor cells (NSC) using a conditional knockout approach driven by nestin-cre. Mice with GCN5-deficient NSC exhibit a 25% reduction in brain mass with a microcephaly phenotype similar to that observed in nestin-cre driven knockouts of...

List of genes upregulated both in GCN5 KO NSC and in N-Myc KO NSC. Genes are listed by name and sorted in descending order by fold change in the GCN5 KO. (PDF)

List of genes downregulated both in GCN5 KO NSC and in N-Myc KO NSC. Genes are listed by name and sorted in descending order by fold change in the GCN5 KO. (PDF)

Introduction The metabolome consists of an immense number of components connected by complex biological pathways spanning a wide array of structural classes such as lipids, carbohydrates, amino acids, and nucleotide structures. This chemical diversity presents a particular challenge for metabolomic study as a single analytical method cannot encompa...

c-Myc is involved in the control of diverse cellular processes and implicated in the maintenance of different tissues including the neural crest. Here, we report that c-Myc is particularly important for pigment cell development and homeostasis. Targeting c-Myc specifically in the melanocyte lineage using the floxed allele of c-Myc and Tyr::Cre tran...

Tables S1 to S5. Table S1. Miz-1 direct targets. Table S2. Myc direct targets. Table S3. Miz-1 and Myc targets. Table S4. Myc knockdown gene expression changes. Table S5. Miz-1 knockdown gene expression changes.

Figure S1. Confirmation of Myc knockdown in human embryonic stem (ES) cells by western blot analysis.

Figure S4. Myc associates with Miz-1, HDACs, and DNMT3A in vivo in human embryonic stem (ES) cells. (A) Coimmunoprecipitation with a c-myc antibody or a non-specific IgG. Western blot analysis was performed using antibodies specific to Miz-1, Dnmt3a, HDAC1, HDAC2, and HDAC3. TRIM28 was used as a negative control for the interaction with Myc. (B) Ch...

Figure S2. Differentiation of human embryonic stem (ES) cells into embryoid bodies (EBs) leads to a drastic reduction of levels of Myc bound to Hox genes, and a significant upregulation of differentiation-associated genes. (A) Chromatin immunoprecipitation (ChIP) analysis of N-Myc and Miz-1 binding in human ES cells and EBs. (B, C) Real-time quanti...

Figure S3. Confirmation of Miz-1 knockdown (KD) in human embryonic stem (ES) cells. (A) Western blot analysis of Miz-1 protein levels. (B) Phase contrast images of human ES cells transduced with either scrambled small hairpin (sh)RNA control or shRNA specific to Miz-1. Images were taken at 4 × magnification.

A proposed role for Myc in maintaining mouse embryonic stem (ES) cell pluripotency is transcriptional repression of key differentiation-promoting genes, but detail of the mechanism has remained an important open topic. To test the hypothesis that the zinc finger protein Miz-1 plays a central role, in the present work we conducted chromatin immunopr...

Induced pluripotent stem cells (iPSCs) hold great promise for autologous cell therapies, but significant roadblocks remain to translating iPSCs to the bedside. For example, concerns about the presumed autologous transplantation potential of iPSCs have been raised by a recent paper demonstrating that iPSC-derived teratomas were rejected by syngeneic...

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Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-divi...

Myc family members play crucial roles in regulating cell proliferation, size, and differentiation during organogenesis. Both N-myc and c-myc are expressed throughout inner ear development. To address their function in the mouse inner ear, we generated mice with conditional deletions in either N-myc or c-myc. Loss of c-myc in the inner ear causes no...

Separate murine knockout (KO) of either c- or N-myc genes in neural stem and precursor cells (NSC) driven by nestin-cre causes microcephaly. The cerebellum is particularly affected in the N-myc KO, leading to a strong reduction in cerebellar granule neural progenitors (CGNP) and mature granule neurons. In humans, mutation of N-myc also causes micro...

Comment on: Parathath SR, et al. Cell Cycle 2010; 9:4013–24.

While endogenous Myc (c-myc) and Mycn (N-myc) have been reported to be separately dispensable for murine embryonic stem cell (mESC) function, myc greatly enhances induced pluripotent stem (iPS) cell formation and overexpressed c-myc confers LIF-independence upon mESC. To address the role of myc genes in ESC and in pluripotency generally, we conditi...

myc genes are associated with a wide variety of human cancers including most types of nervous system tumors. While the mechanisms by which myc overexpression causes tumorigenesis are multifaceted and have yet to be clearly elucidated, they are at least in part related to endogenous myc function in normal cells. Knockout (KO) of either c-myc or N-my...

Murine knockouts (KO) of myc genes have provided substantial insight into not only their normal functions during development, but also shed light on their tumorigenic functions when they are misregulated. c-myc and N-myc genes both are essential in neural stem and precursor cells (NSC) for normal mouse brain growth[1,2]. The fact that mutation of N...

Basal progenitors (also called non-surface dividing or intermediate progenitors) have been proposed to regulate the number of neurons during neocortical development through expanding cells committed to a neuronal fate, although the signals that govern this population have remained largely unknown. Here, we show that N-myc mediates the functions of...

I [Paul Knoepfler] started working with mice as a postdoc at the Fred Hutchinson Cancer Research Center about a decade ago. The lab maintained hundreds of mice and I was responsible for more than 100 of them. The method we used to keep track of the mice was decidedly low tech: a pen and paper. Basic

The activating E2f transcription factors (E2f1, E2f2 and E2f3) induce transcription and are widely viewed as essential positive cell cycle regulators. Indeed, they drive cells out of quiescence, and the 'cancer cell cycle' in Rb1 null cells is E2f-dependent. Absence of activating E2fs in flies or mammalian fibroblasts causes cell cycle arrest, but...

Myc regulates key cellular processes including cell cycle, differentiation, and apoptosis. It has long been thought to direct these functions by acting solely as a classic transcription factor regulating expression of a small number of key target genes through discrete chromatin events in their promoters. A recent wave of genomics studies together...

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Many of the earliest stem cell studies were conducted on cells isolated from tumors rather than from embryos. Of particular interest was research on embryonic carcinoma cells (EC), a type of stem cell derived from teratocarcinoma. The EC research laid the foundation for the later discovery of and subsequent work on embryonic stem cells (ESC). Both...

myc genes are best known for causing tumors when overexpressed, but recent studies suggest endogenous myc regulates pluripotency and self-renewal of stem cells. For example, N-myc is associated with a number of tumors including neuroblastoma, but also plays a central role in the function of normal neural stem and precursor cells (NSC). Both c- and...

Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Alth...

Myc proteins have long been modeled to operate strictly as classic gene-specific transcription factors; however, we find that N-Myc has a robust role in the human genome in regulating global cellular euchromatin, including that of intergenic regions. Strikingly, 90% to 95% of the total genomic euchromatic marks histone H3 acetylated at lysine 9 and...

Mutations that cause autosomal recessive primary microcephaly (MCPH), including MCPH1 through MCPH6, have provided insight into the normal programming that directs brain growth and defines ultimate brain size. The common denominator in these mutations is that they all manifest within neural stem and progenitor cells, decreasing their numbers at var...

Screening cocktails of candidate genes for induction of pluripotency and self-renewal in nonstem cells has identified a surprising new embryonic stem cell regulator, the myc proto-oncogene. Here the possible mechanisms by which myc controls self-renewal and pluripotency are discussed.

Myc family members play crucial roles in regulating cell proliferation, size, differentiation, and survival during development. We found that N-myc is expressed in retinal progenitor cells, where it regulates proliferation in a cell-autonomous manner. In addition, N-myc coordinates the growth of the retina and eye. Specifically, the retinas of N-my...