Biology subjects

Dunlap, J. C.

Publications and source records attributed to Dunlap, J. C..

18 recordsLinked to original sources

bioRxiv2026

SAD-6/ATRX enables broad genome surveillance and defense in fungi

The chromatin remodeler ATRX and its orthologs maintain genome function by regulating repetitive DNA and dynamic chromatin, and their activities have been canonically associated with replication-independent deposition of the histone H3.3 variant. This model is difficult to reconcile with fungi, which encode ATRX orthologs but lack H3 variants that may separately support replication-coupled and replication-independent deposition. Here we show that the fungal ATRX ortholog SAD-6 instead relies on a highly divergent histone H4 variant (H4v) to mediate broad genome surveillance and defense. Deposition of H4v is strictly SAD-6-dependent and thus provides a sensitive genome-wide readout of SAD-6 activity, revealing its functions at telomeres, tRNA and rDNA loci, AT-rich DNA, artificial transgenes, decaying mobile elements, and many genic regions. We further show that SAD-6 is required for a pathway of repeat-induced point mutation (RIP) that also requires DIM-5, a conserved SUV39 methyltransferase that mediates trimethylation of histone H3 lysine-9 in heterochromatin. Together, these findings establish ATRX-like remodelers as broad regulators of genome surveillance and defense in fungi that act through a highly divergent histone H4 variant rather than H3.3. Given that RIP is proposed to recognize repetitive DNA via recombination-independent homologous pairing, the requirement for SAD-6 in RIP suggests that ATRX-like remodelers may couple DNA pairing to heterochromatin nucleation on repeats.

molecular biology↗

bioRxiv2026

Roles for Phosphatase PP4 in Rhythmicity and Compensation in the Neurospora Circadian System

The circadian clock is a highly conserved timer which allows organisms to anticipate future conditions driven by the 24-hour day on planet Earth. Circadian clocks from fungi to mammals are based on a transcription-translation feedback loop (TTFL) molecular architecture. Progressive phosphorylation of core clock proteins can alter both their activity and stability and is required for all known circadian TTFLs. The mechanism for kinase control of circadian period has been extensively studied; however, the mechanism(s) whereby phosphatases alter period remain less studied. Based on the observation that strains of Neurospora crassa lacking phosphatase pp4 display a short circadian period, we investigated regulation of pp4 and its role in the clockworks. In addition to period shortening, loss of pp4 results in a significant loss of both temperature and nutritional compensation, consistent with substrates within the core clock. We identify the clock-relevant PP4 phosphatase holoenzyme as a heterotrimer, and identify two activators of PP4 which regulate circadian period. Biochemical and cell biological analyses suggest that PP4 acts in the nucleus, and a mass spectrometry-based screen identified NCU07414, a member of the HSP40 chaperone system, as a novel PP4 binding partner whose loss results in a dramatically shortened period of the clock when ablated. A model consistent with the data suggests that PP4 may act in opposition to kinases to influence the rate of accumulation of the clock-relevant phosphorylations that determine circadian period.

genetics↗

bioRxiv2026

A Screen To Identify Protein Phosphatases with Roles in Circadian Period, Temperature Compensation and Output in the Neurospora Circadian Clock

The circadian clock is a highly conserved evolutionary advantage which allows organisms to anticipate regular changes in daily environmental conditions. Clocks from fungi to mammals rely on a transcription-translation feedback loop (TTFL) mechanism. Phosphorylation is understood to be a critical regulatory step for maintaining the period of the circadian clock and feedback loop closure. The role of kinases in the Neurospora clock has been examined extensively; however, phosphatases have not been systematically interrogated. By re-examining the Neurospora genome using current informatic tools we identified the 30 genes previously identified as encoding protein phosphatases as well as 13 novel genes, and we assessed the function of the core circadian clock in 39 non-essential phosphatases using a real-time luciferase reporter. We observed both period lengthening and shortening effects, which are not restricted to a single phosphatase family or fold. All but one deletion mutant maintained a rhythmic core clock. In addition, we observed a new temperature compensation defect in the previously studied knockout of phosphatase pph-4, the result of nutritional growth conditions.

genetics↗

bioRxiv2026

The Case for Kinases:A Phosphorylation Driven Model for Circadian Temperature Compensation

Circadian rhythms, [~]24-hour biological cycles, enable organisms to anticipate rhythmic environmental cycles so they can assign proper day and night functions that align with those cycles. Circadian rhythms are defined by their ability to be reset by external cues, their capacity to continue to oscillate in the absence of those cues, and their capacity to maintain the rate of the clock across a range of ambient temperatures, a property known as temperature compensation. In the Neurospora clock, the White Collar Complex (WCC) drives expression of FRQ which nucleates a complex including FRH and CK1a that phosphorylates and thereby represses WCC activity. Work to date has suggested that kinases may be involved in temperature compensation and that in Neurospora the primary target of these is FRQ. Here we investigate the genetic relationship between two clock kinases, Casein Kinase I (ck-1a) and Casein Kinase II (cka), in their regulation of temperature compensation using novel alleles, ck-1aD135G and {Delta}cka. We find that that the clock relies on Casein Kinase I more at cold temperature, but this changes as temperature increases, and the clock relies more on Casein Kinase II at warm temperatures. Using quantitative proteomics on FRQ across temperatures, we find that the FRQ phosphorylation landscape is dependent on temperature and is altered in temperature compensation mutants. This leads to the development of a phosphorylation driven model for temperature compensation, where key temperature compensation specific domains on FRQ are phosphorylated to regulate period length in response to temperature, including by Casein Kinase I and Casein Kinase II.

genetics↗

bioRxiv2026

Cell-Autonomous and Systemic Circadian Regulation of Gene Expression in Adipocytes

Circadian clocks strongly influence adipocyte biology. Studying adipocyte circadian regulation in the absence of organismal cues isolates cell-autonomous clock control, providing insight into mechanisms relevant to metabolic disease. To resolve circadian core biological programs, we deeply sequenced bulk RNA from inguinal-derived, in vitro-differentiated adipocytes (IVDAs) over 2.5 days and compared clock-controlled genes (CCGs) with existing mouse supraclavicular brown (BAT) and epididymal white (eWAT) adipose tissue circadian time-course datasets. Using Phase Set Enrichment Analysis (PSEA), we report 21% of the protein-coding transcriptome is rhythmic in IVDAs. Intrinsic circadian regulation governs key processes in energy metabolism, molecular transport and transcription. Integration with in vivo datasets reveals that BAT and IVDAs exhibit more cohesive rhythmic pathways than does eWAT, clustering around the late-night early-morning transition. To explore how these pathways may be regulated, we reexamined a recent interscapular BAT cistrome dataset. Using IVDA transcription factors that were phase-aligned ([≤]4hr) with in vivo as input, motif enrichment analysis revealed two temporally distinct regulatory programs; an early E-box activator ARNT-family/bHLH-PAS program was enriched for transcriptional regulation, RNA metabolism, and signaling pathways, and a late nuclear receptor-associated program enriched for energy metabolism, phospholipid biosynthesis, mitochondrial function, ECM organization, and nuclear receptor signaling. Overall, we identify novel rhythmic transcripts and define cell-autonomous circadian programs in adipocytes whose timing is further sculpted by systemic cues in vivo. Because obesity is associated with adipocyte hypertrophy and hyperplasia, processes likely influenced by circadian regulation, these findings advance our understanding of clock-controlled adipocyte metabolism and its contribution to metabolic dysfunction.

genomics↗

bioRxiv2026

Rhythmic Nuclear Import Mediated by Importins Regulates the Neurospora Circadian Clock.

Circadian clocks in eukaryotes rely on precisely regulated negative feedback loops to generate daily rhythms. However, the delay mechanisms that extend this structurally simple feedback loop to [~]24 hours are not yet fully understood. In the filamentous fungal model organism Neurospora crassa, the negative arm complex, centered by FREQUENCY (FRQ), must enter the nucleus to repress the White Collar Complex (WCC) and close the feedback loop, but the mechanisms and dynamics of its nuclear transport have remained unresolved. Using long-term live-cell imaging and fluorescence recovery after photobleaching (FRAP), we demonstrate that FRQ nuclear import is an active circadian-regulated process that is fastest early in the subjective day and whose rate progressively decreases as nuclear FRQ approaches peak levels, corresponding to altered direct binding between FRQ and Importin . We further establish that Importin is required for the spatial regulation of FRQ and WCC, as well as the correct timing of Neurospora circadian clock, whereas the nuclear accumulation of non-clock-related free WC-2 doesnt require Importin . Analysis of the three Neurospora Importin {beta} homologs reveals that each of them contributes differently to the circadian clock through pathways beyond FRQ or WCC nuclear import. More specifically, we find a genetic interaction between Imp{beta}3 and the phosphatase PPH-4. Together, these findings indicate that nuclear import is a selective, dynamic, and rate-limiting regulatory step in the fungal circadian clock and reveal both conserved and fungal-specific mechanisms by which importins tune circadian timing.

cell biology↗

bioRxiv2025

Core Clock Protein Subcellular Dynamics Coordinate Local and Global Circadian Control in Syncytia.

Circadian rhythms are pervasive among eukaryotes, and the underlying clocks share a common regulatory architecture - a negative feedback loop. A wealth of genetic and biochemical data underpin current perceptions of circadian oscillators but aspects of their cell biology remain cryptic, especially in syncytial systems. We employed novel microfluidic systems and a blind mutant that retains circadian function to simultaneously track multiple clock components in vivo across circadian cycles, revealing remarkable subcellular and subnuclear dynamics of clock proteins and providing insights into spatiotemporal regulation in a multinucleated system. Despite heterogeneity of clock gene (frq) expression, we find robust cycles in FRQ nuclear localization among all nuclei and document free diffusion of multiple clock components among nuclei. Within nuclei, clock components form distinct, small, highly dynamic nuclear bodies that persist throughout the cycle, occasionally co-localizing for circadian regulatory functions. This rich context of in vivo spatiotemporal information illustrates how separate nuclear clocks ensure synchronous regulation of cellular activities across a macroscopic syncytium.

cell biology↗

bioRxiv2025

A Daily Cycle of White Collar Complex Dephosphorylation Sustains Circadian Rhythmicity in Neurospora

As a photoreceptor, the transcription factor complex WCC acutely activates [~]5% of the genome in response to blue light, while as the circadian positive element in the dark WCC influences expression of about 40% of the transcriptome. Among WCC-regulated genes is frq which is both acutely light-activated through a pLRE and circadian-regulated through a C-box promoter element that is not active in constant light. The complex of FRQ, FRH, and CK1, the FFC, phosphorylates WCC at >95 sites, thereby repressing its activity and closing the circadian feedback loop in the dark. Although FFC has no described role in the light, we validated the expectation that FFC-driven WCC phosphorylation also silences C-box promoters in constant light, thereby confirming two classes of WCC targets, C-box-like that are normally repressed in the light and pLRE-like that remain light-active despite FFC-driven WCC phosphorylation. Genome-wide derepression of C-box-like promoters in frq-null fungi may explain reported non-circadian effects seen in some frq-null fungi including reduced virulence and conidiation. Reanalysis of WCC-mediated circadian activation and repression revealed that, while at dusk most WCC is phosphorylated and repressed, subsequent circadian activation is the result of transient dephosphorylation/derepression of just a small subset of this WCC pool; this small active pool drives expression of FRQ, nucleating the FFC, which rapidly re-phosphorylates the WCC pool to repress it, a phosphorylation/dephosphorylation cycle that can run for days without new WCC synthesis. The realization that both FFC and WCC are regulated primarily through phosphorylation rather than turnover leaves the circadian oscillator looking much like a phoscillator, emphasizing the primacy of post-translational regulation in timekeeping. SignificanceAt the core of circadian clocks of fungi and animals, a protein heterodimer drives expression of gene(s) whose products inactivate the heterodimer via phosphorylation. To sustain the cycle through multiple days, the activity of the heterodimer must be restored, but the means through which this happens have been unclear. In the clock model Neurospora, the White Collar Complex (WCC) is the heterodimer and FFC is the complex that inactivates it. We determined that WCC activity is restored principally by removal of the inhibitory phosphorylations and that for at least several days no new synthesis of WCC is required. The results confirm the existence of a large pool of inactive WCC in the cell and highlights the delicate balance between FCC-dependent phosphorylation/inactivation and phosphatase-dependent dephosphorylation/reactivation. Each morning, this balance allows transient activation of a fraction of the inactive WCC pool, thereby restarting the circadian cycle.

biochemistry↗

bioRxiv2023

Acetylation of WCC is dispensable for the core circadianclock but differentially regulates acute light responses in Neurospora

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency (frq) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) has been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the mono-methylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCCs circadian activity is controlled mainly by phosphorylation. Interestingly, light-induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses.

biochemistry↗

bioRxiv2023

A crucial role for dynamic expression of components encoding the negative arm of the circadian clock

In the Neurospora circadian system, the White Collar Complex (WCC) drives expression of the principal circadian negative arm component frequency (frq). FRQ interacts with FRH (FRQ-interacting helicase) and CK-1 forming a stable complex that represses its own expression by inhibiting WCC. In this study, a genetic screen identified a gene, designated as brd-8, that encodes a conserved auxiliary subunit of the NuA4 histone acetylation complex. Loss of brd-8 reduces H4 acetylation and RNA polymerase (Pol) II occupancy at frq and other known circadian genes, and leads to a long circadian period, delayed phase, and defective overt circadian output at some temperatures. In addition to strongly associating with the NuA4 histone acetyltransferase complex, BRD-8 is also found complexed with the transcription elongation regulator BYE-1. Expression of brd-8, bye-1, histone hH2Az, and several NuA4 subunits is controlled by the circadian clock, indicating that the molecular clock both regulates the basic chromatin status and is regulated by changes in chromatin. Taken together, our data identify new auxiliary elements of the fungal NuA4 complex having homology to mammalian components, which along with conventional NuA4 subunits, are required for timely and dynamic frq expression and thereby a normal and persistent circadian rhythm.

biochemistry↗

bioRxiv2023

Domains Required for FRQ-WCC Interaction within the Core Circadian Clock of Neurospora

In the negative feedback loop composing the Neurospora circadian clock, the core element, FREQUENCY (FRQ) binds with FRH (FRQ-interacting RNA helicase) and Casein Kinase 1 (CK1) to form the FRQ-FRH complex (FFC) which represses its own expression by interacting with and promoting phosphorylation of its transcriptional activators White Collar-1 (WC-1) and WC-2 (together forming the White Collar Complex, WCC). Physical interaction between FFC and WCC is a prerequisite for the repressive phosphorylations, and although the motif on WCC needed for this interaction is known, the reciprocal recognition motif(s) on FRQ remains poorly defined. To address this, FFC-WCC was assessed in a series of frq segmental-deletion mutants, confirming that multiple dispersed regions on FRQ are necessary for its interaction with WCC. Biochemical analysis shows that interaction between FFC and WCC but not within FFC or WCC can be disrupted by high salt, suggesting that electrostatic forces drive the association of the two complexes. As a basic sequence on WC-1 was previously identified as a key motif for WCC-FFC assembly, our mutagenetic analysis targeted negatively charged residues of FRQ leading to identification of three Asp/Glu clusters in FRQ that are indispensable for FFC-WCC formation. Surprisingly, in several frq Asp/Glu-to-Ala mutants that vastly diminish FFC-WCC interaction, the core clock still oscillates robustly with an essentially WT period, indicating that the binding strength between the positive and negative elements in the feedback loop is required for the clock but is not a determinant of the period length.

biochemistry↗

bioRxiv2022

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Circadian clocks are composed of molecular oscillators that pace rhythms of gene expression to the diurnal cycle. Therein, transcriptional-translational negative feedback loops (TTFLs) generate oscillating levels of transcriptional repressor proteins that regulate their own gene expression. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH) and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the White-Collar complex (WCC). A key question concerns how FRQ orchestrates molecular interactions at the core of the clock despite containing little predicted tertiary structure. We present the reconstitution and biophysical characterization of FRQ and the FFC in unphosphorylated and highly phosphorylated states. Site-specific spin labeling and pulse- dipolar ESR spectroscopy provides domain-specific structural details on the full-length, 989- residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino-acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic circadian clocks.

biochemistry↗

bioRxiv2022

Structure-function analysis of 110 phosphorylation sites on the circadian clock protein FRQ identifies clusters determining period length and temperature compensation

In the negative feedback loop driving the Neurospora circadian oscillator, the negative element, FREQUENCY (FRQ), inhibits its own expression by promoting phosphorylation of its heterodimeric transcriptional activators, White Collar-1 (WC-1) and WC-2. FRQ itself also undergoes extensive time-of-day-specific phosphorylation with over 100 phosphosites previously documented. Although disrupting individual or certain clusters of phosphorylation sites has been shown to alter circadian period lengths to some extent, how all the phosphorylations on FRQ control its activity is still elusive. In this study, we systematically investigated the role in period determination of all 110 phosphorylated residues reported on FRQ by mutagenetic and luciferase reporter assays. Surprisingly, robust FRQ phosphorylation is still detected even when 84 phosphosites were eliminated altogether; further mutating another 26 phosphoresidues completely abolished FRQ phosphorylation. To identify phosphoresidue(s) on FRQ impacting circadian period length, series of clustered frq phosphomutants covering all the 110 phosphosites were generated and examined for period changes. When phosphosites in the N-terminal and middle regions of FRQ were eliminated, longer periods were mostly seen while removal of phosphorylation in the C-terminal tail result in extremely short periods, among the shortest reported. Interestingly, abolishing the 11 phosphosites in the C-terminal tail of FRQ does not only result in an extremely short period, but also causes an over-compensated circadian oscillator under a range of physiological temperatures. When different groups of phosphomutations on FRQ were combined intramolecularly, an additive effect was observed as expected; unexpectedly, arrhythmicity resulting from one cluster frq phosphorylation mutants was restored by eliminating phosphorylation at another group of sites, suggesting an epistatic effect between phosphoevents. ImportanceExisting in most eukaryotes, circadian clocks are built based on cell-autonomous, auto-regulatory feedback loops in which negative elements feed back to depress their own expression by repressing the positive elements that drive their synthesis. In Neurospora, the WCC transcription activator drives expression of FRQ, which complexes with FRH and CK1 to repress the DNA-binding activity of WCC by promoting phosphorylation at a group of residues of WCC. The phosphorylation status of FRQ determines the circadian period length, acting independent of effects of phosphorylation on FRQ half-life. Reflecting this dominant role of phosphorylation, FRQ is subject to substantial phosphorylation at over 100 sites in a time-of-day-specific manner. However, how this plethora of phosphoevents on FRQ controls its activity in a circadian cycle is still elusive, and prior work had shown limited effects of individual phosphosite point mutants. In this study, a series of frq mutants targeting multisite phosphorylation within domains of FRQ were generated and analyzed in order to define their roles in period determination. A clear pattern of period altering effects was observed in these frq mutants; certain mutants display strong temperature compensation phenotypes, and interestingly, a novel epistatic relationship on rhythmicity between phosphogroups emerged.

genetics↗

bioRxiv2022

Optimized fluorescent proteins for 4-color and photoconvertible live-cell imaging in Neurospora crassa

Fungal cells are quite unique among life in their organization and structure, and yet implementation of many tools recently developed for fluorescence imaging in animal systems and yeast has been slow in filamentous fungi. Here we present analysis of properties of fluorescent proteins in Neurospora crassa as well as describing genetic tools for the expression of these proteins that may be useful beyond cell biology applications. The efficacy of ten different fluorescent protein tags were compared in a constant context of genomic and intracellular location; six different promoters are described for the assessment of the fluorescent proteins and varying levels of expression, as well as a customizable bidirectional promoter system. We present an array of fluorescent proteins suitable for use across the visible light spectrum to allow for 4-color imaging, in addition to a photoconvertible fluorescent protein that enables a change in the color of a small subset of proteins in the cell. These tools build on the rich history of cell biology research in filamentous fungi and provide new tools to help expand research capabilities.

cell biology↗

bioRxiv2022

A role for gene expression and mRNA stability in nutritional compensation of the circadian clock

Compensation is a defining principle of a true circadian clock, where its approximately 24-hour period length is relatively unchanged across environmental conditions. Known compensation effectors directly regulate core clock factors to buffer the oscillators period length from variables in the environment. Temperature Compensation mechanisms have been experimentally addressed across circadian model systems, but much less is known about the related process of Nutritional Compensation, where circadian period length is maintained across physiologically relevant nutrient levels. Using the filamentous fungus Neurospora crassa, we performed a genetic screen under glucose and amino acid starvation conditions to identify new regulators of Nutritional Compensation. Our screen uncovered 16 novel mutants, and together with 4 mutants characterized in prior work, a model emerges where Nutritional Compensation of the fungal clock is achieved at the levels of transcription, chromatin regulation, and mRNA stability. However, eukaryotic circadian Nutritional Compensation is completely unstudied outside of Neurospora. To test for conservation in cultured mammalian cells, we selected the top two hits from our fungal genetic screen, performed siRNA knockdown experiments of the mammalian homologs, and characterized the cell lines with respect to compensation. We find that the wild-type mammalian clock is also compensated across a large range of external glucose concentrations, as observed in Neurospora, and that knocking down CPSF6 or SETD2 in human cells also results in nutrient-dependent period length changes. We conclude that, like Temperature Compensation, Nutritional Compensation is a conserved circadian process in fungal and mammalian clocks and that it may share common molecular determinants.

genetics↗

bioRxiv2021

Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa.

Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.

cell biology↗

bioRxiv2018

Prediction Interval Ranking Score: Identification of Invariant Expression from Time Series

MotivationIdentification of constitutive reference genes is critical for analysis of gene expression. Large numbers of high throughput time series expression data are available, but current methods for identifying invariant expression are not tailored for time series. Identification of reference genes from these data sets can benefit from methods which incorporate the additional information they provide.\n\nResultsHere we show that we can improve identification of invariant expression from time series by modelling the time component of the data. We implement the Prediction Interval Ranking Score (PIRS) software, which screens high throughput time series data and provides a ranked list of reference candidates. We expect that PIRS will improve the quality of gene expression analysis by allowing researchers to identify the best reference genes for their system from publicly available time series.\n\nAvailabilityPIRS can be downloaded and installed with dependencies using pip install pirs and Python code and documentation is available for download at https://github.com/aleccrowell/PIRS.\n\nContactalexander.m.crowell@gmail.com

bioinformatics↗

bioRxiv2018

Learning and Imputation for Mass-spec Bias Reduction (LIMBR)

MotivationDecreasing costs are making it feasible to perform time series proteomics and genomics experiments with more replicates and higher resolution than ever before. With more replicates and time points, proteome and genome-wide patterns of expression are more readily discernible. These larger experiments require more batches exacerbating batch effects and increasing the number of bias trends. In the case of proteomics, where methods frequently result in missing data this increasing scale is also decreasing the number of peptides observed in all samples. The sources of batch effects and missing data are incompletely understood necessitating novel techniques.\n\nResultsHere we show that by exploiting the structure of time series experiments, it is possible to accurately and reproducibly model and remove batch effects. We implement Learning and Imputation for Mass-spec Bias Reduction (LIMBR) software, which builds on previous block based models of batch effects and includes features specific to time series and circadian studies. To aid in the analysis of time series proteomics experiments, which are often plagued with missing data points, we also integrate an imputation system. By building LIMBR for imputation and time series tailored bias modeling into one straightforward software package, we expect that the quality and ease of large-scale proteomics and genomics time series experiments will be significantly increased.\n\nContactalexander.m.crowell@gmail.com, jay.c.dunlap@dartmouth.edu

bioinformatics↗