The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs.

Translational control is critical for cell fate transitions during development, lineage specification, and tumorigenesis. Here, we show that the transcription factor double homeobox protein 4 (DUX4), and its previously characterized transcriptional program, broadly regulates translation to change the cellular proteome. DUX4 is a key regulator of zygotic genome activation in human embryos, whereas misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and is associated with MHC-I suppression and immune evasion in cancer. We report that translation initiation and elongation factors are disrupted downstream of DUX4 expression in human myoblasts. Genome-wide translation profiling identified mRNAs susceptible to DUX4-induced translation inhibition, including those encoding antigen presentation factors and muscle lineage proteins, while DUX4-induced mRNAs were robustly translated. Endogenous expression of DUX4 in human FSHD myotubes and cancer cell lines also correlated with reduced protein synthesis and MHC-I presentation. Our findings reveal that DUX4 orchestrates cell state conversion by suppressing the cellular proteome while maintaining translation of DUX4-induced mRNAs to promote an early developmental program.

A conserved maternal-specific repressive domain in Zelda revealed by Cas9-mediated mutagenesis in Drosophila melanogaster.

In nearly all metazoans, the earliest stages of development are controlled by maternally deposited mRNAs and proteins. The zygotic genome becomes transcriptionally active hours after fertilization. Transcriptional activation during this maternal-to-zygotic transition (MZT) is tightly coordinated with the degradation of maternally provided mRNAs. In Drosophila melanogaster, the transcription factor Zelda plays an essential role in widespread activation of the zygotic genome. While Zelda expression is required both maternally and zygotically, the mechanisms by which it functions to remodel the embryonic genome and prepare the embryo for development remain unclear. Using Cas9-mediated genome editing to generate targeted mutations in the endogenous zelda locus, we determined the functional relevance of protein domains conserved amongst Zelda orthologs. We showed that neither a conserved N-terminal zinc finger nor an acidic patch were required for activity. Similarly, a previously identified splice isoform of zelda is dispensable for viability. By contrast, we identified a highly conserved zinc-finger domain that is essential for the maternal, but not zygotic functions of Zelda. Animals homozygous for mutations in this domain survived to adulthood, but embryos inheriting these loss-of-function alleles from their mothers died late in embryogenesis. These mutations did not interfere with the capacity of Zelda to activate transcription in cell culture. Unexpectedly, these mutations generated a hyperactive form of the protein and enhanced Zelda-dependent gene expression. These data have defined a protein domain critical for controlling Zelda activity during the MZT, but dispensable for its roles later in development, for the first time separating the maternal and zygotic requirements for Zelda. This demonstrates that highly regulated levels of Zelda activity are required for establishing the developmental program during the MZT. We propose that tightly regulated gene expression is essential to navigate the MZT and that failure to precisely execute this developmental program leads to embryonic lethality.

Transcriptional activation is a conserved feature of the early embryonic factor Zelda that requires a cluster of four zinc fingers for DNA binding and a low-complexity activation domain.

Delayed transcriptional activation of the zygotic genome is a nearly universal phenomenon in metazoans. Immediately following fertilization, development is controlled by maternally deposited products, and it is not until later stages that widespread activation of the zygotic genome occurs. Although the mechanisms driving this genome activation are currently unknown, the transcriptional activator Zelda (ZLD) has been shown to be instrumental in driving this process in Drosophila melanogaster. Here we define functional domains of ZLD required for both DNA binding and transcriptional activation. We show that the C-terminal cluster of four zinc fingers mediates binding to TAGteam DNA elements in the promoters of early expressed genes. All four zinc fingers are required for this activity, and splice isoforms lacking three of the four zinc fingers fail to activate transcription. These truncated splice isoforms dominantly suppress activation by the full-length, embryonically expressed isoform. We map the transcriptional activation domain of ZLD to a central region characterized by low complexity. Despite relatively little sequence conservation within this domain, ZLD orthologs from Drosophila virilis, Anopheles gambiae, and Nasonia vitripennis activate transcription in D. melanogaster cells. Transcriptional activation by these ZLD orthologs suggests that ZLD functions through conserved interactions with a protein cofactor(s). We have identified distinct DNA-binding and activation domains within the critical transcription factor ZLD that controls the initial activation of the zygotic genome.

Facioscapulohumeral Dystrophy: Molecular Basis and Therapeutic Opportunities.

Facioscapulohumeral dystrophy (FSHD) is caused by misexpression of the early embryonic transcription factor Double Homeobox Protein 4 (DUX4) in skeletal muscle. DUX4 is normally expressed at the 4-cell stage of the human embryo and initiates a portion of the first wave of embryonic gene expression that establishes the totipotent cells of the embryo. Following brief expression, the DUX4 locus is suppressed by epigenetic silencing and remains silenced in nearly all somatic cells. Mutations that cause FSHD decrease the efficiency of epigenetic silencing of the DUX4 locus and result in aberrant expression of this transcription factor in skeletal muscles. DUX4 expression in these skeletal muscles reactivates part of the early totipotent program and suppresses the muscle program-resulting in a progressive muscular dystrophy that affects some muscles earlier than others. These advances in understanding the cause of FSHD have led to multiple therapeutic strategies that are now entering clinical trials.

DUX4 expression in cancer induces a metastable early embryonic totipotent program.

The transcription factor DUX4 regulates a portion of the zygotic gene activation (ZGA) program in the early embryo. Many cancers express DUX4 but it is unknown whether this generates cells similar to early embryonic stem cells. Here we identified cancer cell lines that express DUX4 and showed that DUX4 is transiently expressed in a small subset of the cells. DUX4 expression activates the DUX4-regulated ZGA transcriptional program, the subsequent 8C-like program, and markers of early embryonic lineages, while suppressing steady-state and interferon-induced MHC class I expression. Although DUX4 was expressed in a small number of cells under standard culture conditions, DNA damage or changes in growth conditions increased the fraction of cells expressing DUX4 and its downstream programs. Our demonstration that transient expression of endogenous DUX4 in cancer cells induces a metastable early embryonic stem cell program and suppresses antigen presentation has implications for cancer growth, progression, and immune evasion.

Cell-type-specific chromatin occupancy by the pioneer factor Zelda drives key developmental transitions in Drosophila.

During Drosophila embryogenesis, the essential pioneer factor Zelda defines hundreds of cis-regulatory regions and in doing so reprograms the zygotic transcriptome. While Zelda is essential later in development, it is unclear how the ability of Zelda to define cis-regulatory regions is shaped by cell-type-specific chromatin architecture. Asymmetric division of neural stem cells (neuroblasts) in the fly brain provide an excellent paradigm for investigating the cell-type-specific functions of this pioneer factor. We show that Zelda synergistically functions with Notch to maintain neuroblasts in an undifferentiated state. Zelda misexpression reprograms progenitor cells to neuroblasts, but this capacity is limited by transcriptional repressors critical for progenitor commitment. Zelda genomic occupancy in neuroblasts is reorganized as compared to the embryo, and this reorganization is correlated with differences in chromatin accessibility and cofactor availability. We propose that Zelda regulates essential transitions in the neuroblasts and embryo through a shared gene-regulatory network driven by cell-type-specific enhancers.

DUX4-Induced Histone Variants H3.X and H3.Y Mark DUX4 Target Genes for Expression.

The DUX4 transcription factor is briefly expressed in the early cleavage-stage embryo, where it induces an early wave of zygotic gene transcription, whereas its mis-expression in skeletal muscle causes the muscular dystrophy facioscapulohumeral dystrophy (FSHD). Here, we show that DUX4 induces the expression of the histone variants H3.X and H3.Y. We have used a myoblast cell line with doxycycline-inducible DUX4 to show that these histone variants are incorporated throughout the body of DUX4-induced genes. Following a brief pulse of DUX4, these histones contribute to greater perdurance and to enhanced re-activation of DUX4 target gene expression. These findings provide a model for H3.X/Y as a chromatin mechanism that facilitates the expression of DUX4 target genes subsequent to a brief pulse of DUX4 expression.

Regulatory principles governing the maternal-to-zygotic transition: insights from Drosophila melanogaster.

The onset of metazoan development requires that two terminally differentiated germ cells, a sperm and an oocyte, become reprogrammed to the totipotent embryo, which can subsequently give rise to all the cell types of the adult organism. In nearly all animals, maternal gene products regulate the initial events of embryogenesis while the zygotic genome remains transcriptionally silent. Developmental control is then passed from mother to zygote through a process known as the maternal-to-zygotic transition (MZT). The MZT comprises an intimately connected set of molecular events that mediate degradation of maternally deposited mRNAs and transcriptional activation of the zygotic genome. This essential developmental transition is conserved among metazoans but is perhaps best understood in the fruit fly, Drosophila melanogaster. In this article, we will review our understanding of the events that drive the MZT in Drosophila embryos and highlight parallel mechanisms driving this transition in other animals.

An Hdac1/Rpd3-Poised Circuit Balances Continual Self-Renewal and Rapid Restriction of Developmental Potential during Asymmetric Stem Cell Division.

How the developmental potential of differentiating stem cell progeny becomes rapidly and stably restricted following asymmetric stem cell division is unclear. In the fly larval brain, earmuff (erm) uniquely functions to restrict the developmental potential of intermediate neural progenitors (INPs) generated by asymmetrically dividing neural stem cells (neuroblasts). Here we demonstrate that the histone deacetylase Hdac1/Rpd3 functions together with self-renewal transcriptional repressors to maintain the erm immature INP enhancer in an inactive but poised state in neuroblasts. Within 2 hr of immature INP birth, downregulation of repressor activities alleviates Rpd3-mediated repression on the erm enhancer, enabling acetylation of multiple histone proteins and activating Erm expression. Erm restricts the developmental potential in immature INPs by repressing genes encoding neuroblast transcriptional activators. We propose that poising the fast-activating enhancers of master regulators of differentiation through continual histone deacetylation in stem cells enables self-renewal and rapid restriction of developmental potential following asymmetric division.

Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease.

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.

Conjugated metallorganic macrocycles: opportunities for coordination-driven planarization of bidentate, pyridine-based ligands.

Two conjugated systems that can be constrained to planarity via metal coordination have been generated and their metal complexes studied. The potential for these architectures to be incorporated into metal-sensing arylene ethynylene/vinylene oligomers and polymers was probed by verifying that these ligands (1) bind strongly to Ag(I) and Pd(II) cations, and (2) that this event leads to complexes that are planar. Single crystal structures confirm that introduction of Ag(I) or Pd(II) cations enforces planarity in the newly formed macrocycles. Likewise, (1)H-NMR titration studies reveal stoichiometric binding of Pd(II) and strong binding of Ag(I) (K(a (Ligand 1)) = 1.3 × 10(2) M(-1); K(a (Ligand 2)) = 5.4 × 10(2) M(-1)) for each conjugated ligand.