Christopher L. Baker

Post-translational modifications in circadian rhythms

The pace has quickened in circadian biology research. In particular, an abundance of results focused on post-translational modifications (PTMs) is sharpening our view of circadian molecular clockworks. PTMs affect nearly all aspects of clock biology; in some cases they are essential for clock function and in others, they provide layers of regulatory fine-tuning. Our goal is to review recent advances in clock PTMs, help make sense of emerging themes, and spotlight intriguing (and perhaps controversial) new findings. We focus on PTMs affecting the core functions of eukaryotic clocks, in particular the functionally related oscillators in Neurospora crassa, Drosophila melanogaster, and mammalian cells.

Quantitative Proteomics Reveals a Dynamic Interactome and Phase-Specific Phosphorylation in the Neurospora Circadian Clock

Circadian systems are comprised of multiple proteins functioning together to produce feedback loops driving robust, approximately 24 hr rhythms. In all circadian systems, proteins in these loops are regulated through myriad physically and temporally distinct posttranslational modifications (PTMs). To better understand how PTMs impact a circadian oscillator, we implemented a proteomics-based approach by combining purification of endogenous FREQUENCY (FRQ) and its interacting partners with quantitative mass spectrometry (MS). We identify and quantify time-of-day-specific protein-protein interactions in the clock and show how these provide a platform for temporal and physical separation between the dual roles of FRQ. Additionally, by unambiguously identifying over 75 phosphorylated residues, following their quantitative change over a circadian cycle, and examining the phenotypes of strains that have lost these sites, we demonstrate how spatially and temporally regulated phosphorylation has opposing effects directly on overt circadian rhythms and FRQ stability.

The circadian clock of Neurospora crassa.

Circadian clocks organize our inner physiology with respect to the external world, providing life with the ability to anticipate and thereby better prepare for major fluctuations in its environment. Circadian systems are widely represented in nearly all major branches of life, except archaebacteria, and within the eukaryotes, the filamentous fungus Neurospora crassa has served for nearly half a century as a durable model organism for uncovering the basic circadian physiology and molecular biology. Studies using Neurospora have clarified our fundamental understanding of the clock as nested positive and negative feedback loops regulated through transcriptional and post-transcriptional processes. These feedback loops are centered on a limited number of proteins that form molecular complexes, and their regulation provides a physical explanation for nearly all clock properties. This review will introduce the basics of circadian rhythms, the model filamentous fungus N. crassa, and provide an overview of the molecular components and regulation of the circadian clock.

Health and population effects of rare gene knockouts in adult humans with related parents

Rare gene knockouts in adult humans On average, most peoples genomes contain approximately 100 completely nonfunctional genes. These loss-of-function (LOF) mutations tend to be rare and/or occur only as a single copy within individuals. Narasimhan et al. investigated LOF in a Pakistani population with high levels of consanguinity. Examining LOF alleles that were identical by descent, they found, as expected, an absence of homozygote LOF for certain protein-coding genes. However, they also identified many homozygote LOF alleles with no apparent deleterious phenotype, including some that were expected to confer genetic disease. Indeed, one family had lost the recombination-associated gene PRDM9. Science, this issue p. 474 The total loss of protein-coding genes, even those with the potential to confer genetic diseases, can be tolerated. Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3222 British adults of Pakistani heritage with high parental relatedness, discovering 1111 rare-variant homozygous genotypes with predicted loss of function (knockouts) in 781 genes. We observed 13.7% fewer homozygous knockout genotypes than we expected, implying an average load of 1.6 recessive-lethal-equivalent loss-of-function (LOF) variants per adult. When genetic data were linked to the individuals’ lifelong health records, we observed no significant relationship between gene knockouts and clinical consultation or prescription rate. In this data set, we identified a healthy PRDM9-knockout mother and performed phased genome sequencing on her, her child, and control individuals. Our results show that meiotic recombination sites are localized away from PRDM9-dependent hotspots. Thus, natural LOF variants inform on essential genetic loci and demonstrate PRDM9 redundancy in humans.

A Role for Casein Kinase 2 in the Mechanism Underlying Circadian Temperature Compensation

Temperature compensation of circadian clocks is an unsolved problem with relevance to the general phenomenon of biological compensation. We identify casein kinase 2 (CK2) as a key regulator of temperature compensation of the Neurospora clock by determining that two long-standing clock mutants, chrono and period-3, displaying distinctive alterations in compensation encode the beta1 and alpha subunits of CK2, respectively. Reducing the dose of these subunits, particularly beta1, significantly alters temperature compensation without altering the enzymes Q(10). By contrast, other kinases and phosphatases implicated in clock function do not play appreciable roles in temperature compensation. CK2 exerts its effects on the clock by directly phosphorylating FREQUENCY (FRQ), and this phosphorylation is compromised in CK2 hypomorphs. Finally, mutation of certain putative CK2 phosphosites on FRQ, shown to be phosphorylated in vivo, predictably alters temperature compensation profiles effectively phenocopying CK2 mutants.

A Circadian Clock in Neurospora: How Genes and Proteins Cooperate to Produce a Sustained, Entrainable, and Compensated Biological Oscillator with a Period of about a Day

Neurospora has proven to be a tractable model system for understanding the molecular bases of circadian rhythms in eukaryotes. At the core of the circadian oscillatory system is a negative feedback loop in which two transcription factors, WC-1 and WC-2, act together to drive expression of the frq gene. WC-2 enters the promoter region of frq coincident with increases in frq expression and then exits when the cycle of transcription is over, whereas WC-1 can always be found there. FRQ promotes the phosphorylation of the WCs, thereby decreasing their activity, and phosphorylation of FRQ then leads to its turnover, allowing the cycle to reinitiate. By understanding the action of light and temperature on frq and FRQ expression, the molecular basis of circadian entrainment to environmental light and temperature cues can be understood, and recently a specific role for casein kinase 2 has been found in the mechanism underlying circadian temperature-compensation. These data promise molecular explanations for all of the canonical circadian properties of this model system, providing biochemical answers and regulatory logic that may be extended to more complex eukaryotes including humans.

PRDM9 binding organizes hotspot nucleosomes and limits Holliday junction migration

In mammals, genetic recombination during meiosis is limited to a set of 1- to 2-kb regions termed hotspots. Their locations are predominantly determined by the zinc finger protein PRDM9, which binds to DNA in hotspots and subsequently uses its SET domain to locally trimethylate histone H3 at lysine 4 (H3K4me3). This sets the stage for double-strand break (DSB) formation and reciprocal exchange of DNA between chromatids, forming Holliday junctions. Here we report genome-wide analyses of PRDM9-dependent histone modifications using two inbred mouse strains differing only in their PRDM9 zinc finger domain. We show that PRDM9 binding actively reorganizes nucleosomes into a symmetrical pattern, creating an extended nucleosome-depleted region. These regions are centered by a consensus PRDM9 binding motif, whose location and identity was confirmed in vitro. We also show that DSBs are centered over the PRDM9 binding motif within the nucleosome-depleted region. Combining these results with data from genetic crosses, we find that crossing-over is restricted to the region marked by H3K4me3. We suggest that PRDM9-modified nucleosomes create a permissible environment that first directs the location of DSBs and then defines the boundaries of Holliday junction branch migration.

PRDM9 drives evolutionary erosion of hotspots in Mus musculus through haplotype-specific initiation of meiotic recombination.

Meiotic recombination generates new genetic variation and assures the proper segregation of chromosomes in gametes. PRDM9, a zinc finger protein with histone methyltransferase activity, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we measured activity of the Prdm9 Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9Cst arose, and M.m. domesticus, into which Prdm9Cst was introduced experimentally. Comparing these two strains, we find that haplotype differences at hotspots lead to qualitative and quantitative changes in PRDM9 binding and activity. Using Mus spretus as an outlier, we found most variants affecting PRDM9Cst binding arose and were fixed in M.m. castaneus, suppressing hotspot activity. Furthermore, M.m. castaneus×M.m. domesticus F1 hybrids exhibit novel hotspots, with large haplotype biases in both PRDM9 binding and chromatin modification. These novel hotspots represent sites of historic evolutionary erosion that become activated in hybrids due to crosstalk between one parents Prdm9 allele and the opposite parents chromosome. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion.

Decoupling circadian clock protein turnover from circadian period determination

Defining necessary circadian clock elements The circadian clock in organisms as diverse as fungi and humans have a rather similar structure: Timing depends on daily cycles of transcription in circuits in which feedback loops control the timing of oscillations. A critical role has been ascribed to negative elements, which lead to inhibition of their own transcription, and to degradation of these elements, which is signaled by phosphorylation events. However, Larrando et al. show that in the fungus Neurospora, after manipulations that prevent phosphorylation-signaled degradation of the negative element FREQUENCY (FRQ), rhythms still persist (see the Perspective by Kramer). They suggest a model in which other phosphorylation events on Frq (of which there are over 100) must have critical roles in controlling the clock, independent of negative element degradation. Science, this issue 10.1126/science.1257277; see also p. 476 Control of negative circadian elements in the simple fungus Neurospora goes beyond targeted proteolysis. [Also see Perspective by Kramer] INTRODUCTION Circadian oscillators allow individual organisms to coordinate metabolism with day/night cycles and to anticipate such changes. Such oscillators in fungi and animals share a common regulatory architecture centered on transcription and translation-based negative feedback loops. Within such oscillators, extensive coordinated and progressive phosphorylation of negative element proteins leads to their proteasome-mediated degradation. Current clock models posit that this turnover event is the final essential step in the loop and that the time taken to achieve phosphorylation and turnover determines the speed of the circadian clock. The clock in Neurospora exemplifies such oscillators: FREQUENCY (FRQ) is a negative element, and its half-life is well correlated with circadian period length. Surprisingly, however, using real-time reporters in cells with compromised proteasomal turnover, we unveiled an unexpected uncoupling between negative element half-life and circadian period determination. RATIONALE We followed FRQ dynamics as well as transcriptional activity of the frq promoter in vivo using luciferase-based reporters. FRQ turnover was tracked through Western blotting, and kinase inhibitors helped to test the correlation between phosphorylation and period length. Strains bearing frq alleles causing abnormal period lengths were used, as were strains with diminished FRQ turnover, including knockouts of both the F-box protein FWD-1 (a ubiquitin ligase that mediates FRQ proteasomal degradation) and individual components of the COP9 signalosome. RESULTS Without FWD-1, FRQ turnover is severely compromised and circadian regulation of development is lost; however, in such Δfwd-1 cells, the amount of FRQ still oscillated, the result of cyclic transcription of frq and reinitiation of FRQ synthesis. The circadian nature of these rhythms was confirmed by examining well-established frq mutants having altered periods. Analyses of additional strains bearing knockouts of individual COP9 signalosome components further confirmed circadian oscillations in FRQ amounts, despite compromised FRQ turnover. Broadly accepted oscillator models posit that negative element stability determines clock period length; thus, Δfwd-1 strains with long FRQ half-lives are predicted to have extremely long periods. This, however, is not seen: Period is mainly determined by the characteristics of the frq allele irrespective of the half-life of this negative element. Partial inhibition of overall phosphorylation provided additional evidence that clock protein phosphorylation events, not the resulting stability changes, provide key information in determining period length. DISCUSSION The long-standing and assumed causal loop uniting clock protein phosphorylation, stability, and period determination should be revisited. Data indicate that qualities of FRQ—in particular, its phosphorylation status rather than its quantity—are crucial for determining when the circadian feedback loop is completed and can be restarted. Previously described strong correlations between clock protein phosphorylation and half-life and between half-life and period length are, in fact, just correlations that do not always imply cause and effect. Although degradation is the final outcome of FRQ posttranslational modifications, phosphorylation and its effects of secondary, tertiary, and quaternary protein structure may actually be the key elements determining clock speed. Although it may be premature to broadly generalize these findings to all circadian oscillators, diverse data from several animal circadian systems are not inconsistent with this revised model. Distinct roles for FRQ phosphorylation and degradation in the clock. White Collar-1 and -2 (WC-1 and WC-2) activate frq expression and FRQ (with FRH and CK1) later inhibit expression. FRQ phosphorylation affects interactions with WC-1/WC-2, reducing inhibition. By influencing these key interactions, FRQ phosphorylations determine the rate at which core clock events, those within the clock face, occur. After key phosphorylations close the loop, degradation-related events need not affect circadian period. The mechanistic basis of eukaryotic circadian oscillators in model systems as diverse as Neurospora, Drosophila, and mammalian cells is thought to be a transcription-and-translation–based negative feedback loop, wherein progressive and controlled phosphorylation of one or more negative elements ultimately elicits their own proteasome-mediated degradation, thereby releasing negative feedback and determining circadian period length. The Neurospora crassa circadian negative element FREQUENCY (FRQ) exemplifies such proteins; it is progressively phosphorylated at more than 100 sites, and strains bearing alleles of frq with anomalous phosphorylation display abnormal stability of FRQ that is well correlated with altered periods or apparent arrhythmicity. Unexpectedly, we unveiled normal circadian oscillations that reflect the allelic state of frq but that persist in the absence of typical degradation of FRQ. This manifest uncoupling of negative element turnover from circadian period length determination is not consistent with the consensus eukaryotic circadian model.

The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo

In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we suggest that it may play the same role in meiosis.