bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451963; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Native MOWChIP-seq: Genome-wide profiles of key protein bindings reveal functional differences among various brain regions
Zhengzhi Liu1, Lynette B. Naler2, Yan Zhu2, Chengyu Deng2, Qiang Zhang2, Bohan Zhu2, Zirui Zhou2, Mimosa Sarma2, Alexander Murray3, Hehuang Xie3, Chang Lu2,*
1 Department of Biomedical Engineering and Mechanics, Virginia Tech, Blacksburg, VA 2 Department of Chemical Engineering, Virginia Tech, Blacksburg, VA 3 Department of Biomedical Sciences & Pathobiology, Virginia Tech, Blacksburg, VA Corresponding author: [email protected]
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Abstract
Genome-wide profiling of interactions between genome and various functional proteins is
critical for understanding regulatory processes involved in development and diseases.
Conventional assays require a large number of cells and high-quality data on tissue samples are
scarce. Here we optimized a low-input chromatin immunoprecipitation followed by sequencing
(ChIP-seq) technology for profiling RNA polymerase II (Pol II), transcription factor (TF), and
enzyme binding at the genome scale. The new approach, termed native MOWChIP-seq,
produces high-quality binding profiles using 1000-50,000 cells. We used the approach to
examine the binding of Pol II and two TFs (EGR1 and MEF2C) in cerebellum and prefrontal
cortex of mouse brain and found that their binding profiles are highly reflective of the functional
differences between the two brain regions. Our analysis reveals the potential for linking genome-
wide TF or Pol II profiles with neuroanatomical origins of brain cells.
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Introduction
Protein-DNA interactions are widely and critically involved in the regulations of gene
transcription and expression1. RNA polymerase II (Pol II) transcribes protein-coding genes into
mRNA following several steps: formation of preinitiation complex, promoter-proximal pausing,
elongation and termination. Pol II binding occurs throughout the genome, with enrichment at
regions being either actively expressed or readied for imminent transcription upon environmental
cues2,3. This latter phenomenon of Pol II pausing is the rate limiting step on more than 70% of
metazoan genes and has been shown to play key biological roles.3-6 Similarly, transcription
factors (TFs) bind to DNA or cofactors and participate in gene regulations in a sequence-specific
manner.1,7 TFs control key aspects of cellular biology including cell differentiation, development
patterning, and immune response. 8,9 Finally, enzymes such as histone acetyl transferases (HATs)
and histone deacetylases (HDACs) closely interact with the genome to effect epigenetic
modifications (histone acetylations) that are critically involved in gene silencing and activation10.
For example, HDAC2 binding and activity are involved in memory formation and synaptic
plasticity in brain11. Genome-wide binding profiles of these proteins are highly indicative of
genes being activated or readied in a process. Difference in the binding profile among various
tissue samples reveals underlying genome-wide molecular dynamics and variations involved in
development or disease.
Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is a simple and
direct approach to profile in vivo genome-wide binding of proteins. Although ChIP-seq has been
generating reliable and high-quality data on histone modifications (i.e. the interaction between a
modification histone and the genome)12-14, ChIP-seq results on other types of protein-DNA
interactions tend to be much more challenging. Compared to the robust histone-DNA interaction,
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the interaction between DNA and other protein molecules such as Pol II, TFs, and enzymes may
be harder to preserve even after treatment such as formaldehyde crosslinking. Thus conventional
ChIP-seq requires a large quantity of starting material (>107 cells per assay) for probing genomic
binding of proteins that are not histones and the results tend to have lower reproducibility
compared to histone ChIP-seq.15 These limitations makes it impractical to examine animal and
human tissue samples available only in small quantities. In recent years, significant efforts have
been made in developing low-input ChIP-seq methods12-14,16-22. However, the vast majority of
these methods were only been demonstrated on examining histone modifications.
As an alternative to ChIP, IP-free technologies such as CUT&RUN23,24 and ChIL-seq25
were developed to profile factor binding to the genome. CUT&RUN maps Pol II and TF binding
by cutting and releasing DNA fragments that interact with antibody-targeted Pol II and TFs into
supernatant, requiring as few as 1,000 cells 23,24. The latest variation of CUT&RUN
(CUT&Tag)26 have been used to profile single cells. However, CUT&RUN requires
immobilization of cells to bead surface via concanavalin A and glycolipid interaction and
CUT&RUN data appear to exhibit lower correlation with the gold-standard ChIP-seq data (e.g.
ENCODE data) compared to low-input ChIP-seq technologies 23,24. CUT&RUN datasets may
also be contaminated by DNA sequences that are from unknown sources associated with the
27 process (e.g. sequences containing (TA)n) .
Here we establish micrococcal nuclease (MNase)-digestion-based native MOWChIP-seq
as a general tool for low-input profiling of protein binding to genome. MNase digestion was
previously applied in native ChIP to probe mostly histone modifications28 and also non-histone
proteins including pol II and TFs29-33. However, these studies were all conducted using a large
number of cells (107-108). Our approach, referred to as native MOWChIP-seq or nMOWChIP-
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seq, combines MNase digestion of native chromatin with a microfluidics-based low-input ChIP-
seq technology (MOWChIP-seq12,14) that was previously applied to examine histone
modifications only. We show that MNase digestion is effective for preserving the links between
protein and genome and facilitating low-input profiling. We generated high-quality ChIP-seq
data using as few as 1,000 cells for studying Pol II, 5,000 cells for TF EGR1, and 50,000 cells
for HDAC2. We applied this method to study genome-wide binding of Pol II, EGR1 and MEF2C
in two functional regions of the mouse brain: prefrontal cortex (PFC) and cerebellum. Extensive
variations in RNA Pol II and TF binding were identified between these two brain regions and
these profiles reveal the involvement of these regulatory molecules in the functional difference.
Results
Profiling genome-wide binding of RNA Pol II, TFs, and enzymes
RNA Pol II, TF or enzyme binding has been conventionally studied after crosslinking
using reagent such as formaldehyde that firmly immobilizes the protein to the genomic DNA
34,35. However, our initial results showed that even using our low-input MOWChIP-seq
technology which has proven high efficiency for collecting DNA-protein complexes12,14, low-
quality results were yielded when 100,000 cells were crosslinked and sonicated to create the
chromatin fragments before Pol II ChIP (Supplementary Fig. S1). 54,000 and 17,000 peaks were
yielded in the two technical replicates, compared to 115,000 and 141,000 peaks generated by
nMOWChIP-seq using 10,000 cells per assay. Crosslinking and subsequent sonication may
damage RNA Pol II’s ability to form antigen-antibody complex and seriously limits the ChIP’s
efficiency for collecting targeted fragments.
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We then applied nMOWChIP-seq to profile protein binding to the genome. MNase
digestion (or native ChIP) had primarily been applied to studies associated with histone
modifications where the strong interaction between genomic DNA and histone guarantees the
pulldown of DNA without crosslinking 13,23,36. In our process (Fig. 1A), tissues were first
mechanically homogenized to extract nuclei and cultured cells were directly used. Nuclei or cells
were lysed and digested with MNase to yield chromatin fragments with size range appropriate
for ChIP (150-600 bp). That was followed by the MOWChIP process12,14. Briefly, in a
microfluidic chamber with a partially closed sieve valve, antibody-coated magnetic IP beads
were packed into a dense bed. All chromatin fragments were forced through the bead bed,
drastically increasing the adsorption efficiency of targeted fragments. Oscillatory washing was
then applied to remove non-specific binding, and the beads with bound chromatins were
transferred out of the chamber for DNA elution, library preparation and sequencing.
Using cell line GM12878, we show that our method can generate high quality ChIP-seq
data with input as little as 1,000 cells per assay for Pol II (phospho S5) (Fig. 1B). Pearson
correlations between technical replicates were 0.87, 0.84, 0.60, and 0.54 for 50,000-, 10,000-,
2,000-, and 1,000-cell samples on RNA Pol II, respectively (Figure 1C). We benchmarked our
data against ENCODE data obtained using conventional ChIP-seq method and 20 million cells
per assay. We examined the number of peaks called and the fraction of reads in peaks (FRiP) for
data quality (Supplementary Table S1). The nMOWChIP-seq Pol II data produced averagely
137,000, 128,000, 65,000 and 27,000 peaks with 50,000, 10,000, 2,000 and 1,000 cells per assay,
respectively, compared to 110,000 peaks from the ENCODE data obtained using 20 million
cells. FRiP measures the amount of background in the data and nMOWChIP-seq Pol II data
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showed a decrease from 49.8% to 9.4% with cells per assays decreasing from 50,000 to 1,000,
far exceeding the 1% threshold recommended by ENCODE37.
We also determined that the binding of Pol II was stable enough to be preserved at -
80 °C, a unique property not observed in histone modifications or TFs (Supplementary Figure
S2). From our testing, MNase-digested chromatin can be frozen at -80 °C or on dry ice for 2 d
without substantial degradation in data quality for Pol II.
We profiled a transcription factor, early growth response protein 1 (EGR1), using as few
as 5,000 GM12878 cells (Fig. 1B and 1C, Supplementary Table S1). Pearson correlation
coefficients between the technical replicates were 0.87, 0.87, 0.63, and 0.67 for 100,000-,
50,000-, 10,000-, and 5,000-cell samples, respectively (Fig. 1C). EGR1 data generally show
lower peak numbers and FRiP than Pol II data, as shown by both nMOWChIP-seq and ENCODE
data. FRiP ranged from 8.4% to 3.2% in our nMOWChIP-seq EGR1 data. We also profiled
another TF MEF2C (Supplementary Table S1). A large percentage of the EGR1 (85%) and
MEF2C (75%) peaks overlap with Pol II peaks (Supplementary Figure S3).
Finally, we applied nMOWChIP-seq to examine the binding of histone deacetylase
HDAC2 in GM12878 cells (Fig. 1B and 1C, Supplementary Table S1). At least 50,000 cells
were required to generate good quality data on HDAC2 binding. An average of 1,394 (FRiP =
1.2%) and 8527 peaks (FRiP = 3.1%) were generated using 50,000 and 100,000 cells per assay,
compared to 1820 peaks of ENCODE data obtained using 10 million cells (FRiP = 0.3%). We
also examined HDAC2 binding in mouse brain cells (Supplementary Fig. S4). 100,000 mixed
nuclei and 800,000 FACS-sorted NeuN+ neuronal nuclei were used in each assay and an average
of 3193 (FRiP ~13.1%) and 2112 (FRiP ~10.4%) peaks were produced (Supplementary Table
S1).
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Comparison of Pol II and Pol II-S5 binding profiles
We used two antibodies to differentiate the binding of phosphor-5 activated subset of Pol
II (Pol II-S5, ab5131) and all Pol II regardless of its phosphorylation status (Pol II-total, ab817,
clone 8WG16). Our data show the distinction (Fig. 2). The two profiles on GM12878 cells are
similar in a large fraction of the genome (Fig. 2A). However, some genes (ACTG1 and MDM2
as examples) with Pol II binding over the entire gene body show that Pol II-total profile captures
the initial binding of unphosphorylated Pol II at TSS, which is absent in Pol II-S5 profile in
comparison (Fig. 2B). During the course of binding to a gene, Pol II is unphosphorylated during
the pre-initiation stage, but undergoes phosphorylation once a short (20-60 bp) mRNA begins to
transcribe.2 Being able to differentiate the distinct forms of Pol II is important when the exact Pol
II binding status on specific genes is of interest (e.g. whether pre-initiation or activated Pol II
dominates pausing at a TSS).
To determine whether Pol II-total or Pol II-S5 was more effective in predicting TF
binding sites, we analyzed the overlap of either Pol II-total peaks or Pol II-S5 peaks with EGR1
peaks at varying peak-calling thresholds, shown as receiver operating characteristic (ROC)
curves that display the true positive rate (TPR) versus the false positive rate (FPR) (Fig. 2c). To
quantify the predictive quality, we calculated the area under the curve (AUC) for each of the
ROC curves using our EGR1 binding data as the gold standard. We found that Pol II-S5 profile
had a higher predictive value than Pol II-total (0.899 vs 0.845 using nMOWChIP-seq data, and
0.871 vs 0.807 using ENCODE data). We also tested if Pol II-S5 was more robust than Pol II-
total with samples of lower quality (Fig. 2D). For this, we subsampled our EGR1 data from 25%
to 95% of the original reads, with 5 replicates at each sampling. We then calculated the average
Matthew’s correlation coefficient (MCC), a robust single value quantifier of classifier quality, of
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the five replicates at each subsampling percentage. Pol II-S5 outperformed Pol II-total, both in
our data and in ENCODE’s, regardless of TF data quality.
Differential RNA Pol II binding in prefrontal cortex and cerebellum of mouse brain
We applied nMOWChIP-seq to profile Pol II binding in mouse PFC and cerebellum. PFC
have roles in cognitive functions, decision-making and short-term memory,38,39 while cerebellum
controls motor functions and coordination.40 We reasoned that the functional difference between
the two regions should reflect on the binding of Pol II and TFs that have recognized roles in the
brain. There have not been published ChIP-seq data confirming this.
We mapped Pol II-total binding in B6 mouse brain using nuclei extracted from PFC and
cerebellum (Fig. 3). The genome-wide Pol II profiles were substantially different between
cerebellum and PFC with Pearson’s correlation being 0.67, compared to an average of 0.96
between technical replicates (Fig. 3A and 3B). DiffBind analysis identified 3021 peaks with
higher levels of Pol II binding in PFC than in cerebellum, and 1197 peaks having higher binding
in cerebellum (fold change > 2, p < 10-5). Gene ontology (GO) analysis of these regions showed
that the regions with high Pol II binding intensity in PFC were enriched in terms associated with
memory, learning and anxiety-related response (Fig. 3C). Synaptic plasticity, one of the
fundamentals of learning and memory, was also enriched along with its key components: long
term potentiation and depression (Figure 3C).41 In contrast, the genomic regions with Pol II
binding higher in cerebellum were enriched in terms that were specific to cerebellum (e.g.
cerebellum morphology and development) (Fig. 3C).
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We also examined the Pol II pausing index for the Pol II-bound genes in PFC and
cerebellum. Pol II pausing index (PI) refers to the ratio of Pol II read density between promoter
proximal region (-30 to +300 bp of TSS) and gene body (+300 bp of TSS to TES), with higher PI
indicating more Pol II binding near TSS2,42. Pol II-bound genes can be divided into three
categories based on the PI value: non-paused and expressed (PI<2), paused and expressed
(2
participate in rodent brain development and cell proliferation,43 and Npas4, which is involved in
regulating reward-related learning and memory,44 both showed dramatically higher PI values in
cerebellum than in PFC (Fig. 3D). The data revealed that they were actively expressed in PFC
but paused without expression in cerebellum. In contrast, Slc46a1 and Rsph9 showed higher PI
values in PFC than cerebellum. While both were being expressed in the two tissues, there was
significant pausing of Pol II at TSS in PFC. This suggests that PFC had more potential in
transcribing these genes at a short notice, such as Slc46a1, which encodes a facilitative carrier
for folate.45 Furthermore, we analyzed the distribution of the PI in PFC and cerebellum (Fig. 3E).
36% of genes were being actively expressed in PFC (PI<20), compared to only 25% in
cerebellum. Mouse brain displays more pausing and less active transcription compared to
GM12878 (52% actively expressed genes, Supplementary Fig. S5). This was also within our
expectation because GM12878 cell line was maintained in log phase and actively dividing,
unlike brain cells in adult mice. We identified Pol II-bound genes that displayed significantly
different pausing indexes between PFC and cerebellum (fold change > 3, minimal read density >
0.02 read/bp) (Supplementary Table S2). Among these, Igfbp6 is involved in myelin formation
during central nervous system (CNS) development,46 and Slc1a2 encodes excitatory amino acid
transporter 2, responsible for reuptake of 90% glutamate in CNS.47 Shank3 belongs to the Shank
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gene family that plays a role in synapse formation,48 and Flrt2 encodes a member of the FLRT
protein family that is shown to regulate signaling during mouse development.49
Differential EGR1 and MEF2c binding in prefrontal cortex and cerebellum of mouse brain
We also examined EGR1 and MEF2C (myocyte enhancer factor-2 C) binding using
nuclei extracted from mouse PFC and cerebellum. Chromatin from 100,000 nuclei were used in
each assay, yielding high quality data that revealed differential binding between the two regions
of brain (Figure 4A). We picked several genes as examples (Fig. 4A). Kalrn (Kalirin) plays
important roles in nerve growth50. Higher EGR1/MEF2C activity in PFC was observed on
Gria1(Glutamate receptor 1) which is involved in synaptic transmission.51 Cacna1a is involved in
movement disorder52 and expression of Nfix can influence neural stem cell differentiation.53 Zic1
and Zic4 belong to the family of Zinc finger of the cerebellum (ZIC) protein family,54 whose loss
of function can lead to Dandy-Walker malformation and incomplete cerebellar vermis55. Higher
EGR1 binding on these four genes were seen in cerebellum than in PFC. A large fraction of
EGR1 and MEF2C peaks (68-89%) appear to overlap with Pol II peaks, in both cerebellum and
PFC (Supplementary Fig. S6). We observed correlated EGR1 and MEF2C profiles, with an
average of Pearson’s correlation of 0.74 between the two TFs in PFC and 0.84 in cerebellum
(Fig. 4B). Such correlations were much higher than the one observed in GM12878 cells (r
~0.52). On the other hand, EGR1 and MEF2C present very different profiles between PFC and
cerebellum, with the average correlation of 0.51 between the two brain regions for both TFs. We
further examined the binding difference between PFC and cerebellum for these two TFs. Their
binding sites were much more heavily situated at promoters in PFC (79%) than in cerebellum
(60%) (Fig. 4C). We further analyzed the data using DiffBind to identify regions with
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significantly different level of EGR1/MEF2C binding between PFC and cerebellum.56 In total,
DiffBind identified 1026 peaks with higher EGR1 binding in cerebellum than in PFC, and 1563
peaks with higher MEF2C binding in cerebellum than in PFC (fold change > 2, p < 10-5). These
regions were further analyzed for GO term enrichment analysis using GREAT and linked to
walking behavior, motor coordination, and cerebellum morphology and development in the case
of EGR1, cerebellar development and limb coordination in the case of MEF2C (Figure 4D). In
contrast, very few differential peaks (12 for EGR1 and 1 for MEF2C) were found to have higher
binding signal in PFC than in cerebellum and no GO terms were found.
Discussion
ChIP-seq profiling of proteins bound to the genome is generally much more challenging
than that of modified histones. Histone ChIP-seq can be conducted with crosslinking and
sonication or under native ChIP condition. Due to the robust interaction between histones and
genome, native ChIP-seq for histone modifications can have very high efficiency. In contrast,
previous ChIP-seq of protein bindings often involves crosslinking and sonication that
immobilizes the protein to the interacting DNA sequence before breaking chromatin into
fragments. Crosslinking is often considered necessary when TF, Pol II and enzyme interaction
with the genome is studied, due to perceived needs and benefits for preservation of such
interactions by crosslinking. However, crosslinking and sonication potentially cause epitope
masking57 and damage, respectively, and both affect antibody-antigen interaction critically
involved in ChIP assays. Furthermore, crosslinking may also create artifact peaks at highly
transcribed regions due to protein-protein crosslinking58.
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In this work, by conducting nMOWChIP-seq without crosslinking and sonication, we
demonstrate a low-input technology that works with as few as 1,000-50,000 cells per assay for
profiling a wide range of protein-genome interactions. We show that nMOWChIP-seq method
effectively preserves the interaction between Pol II/TFs/enzyme and the genome. In our
approach, the required number of cells depends on the robustness of the protein binding to the
genome under native ChIP conditions, the number of binding sites, and the quality of the
antibody. Compared to the state-of-the-art ChIP-seq data taken using millions of cells per assay,
our datasets generally show very high signal-to-noise ratio and low background, which are
characterized by high FRiP values. Our method is also comparable to CUT&RUN24,27 in terms of
data quality.
Compared to histone modification data, ChIP-seq data on Pol II, TFs, enzyme in tissues
are very scarce. We applied nMOWChIP-seq to profile Pol II, and key TFs EGR1 and MEF2C in
a brain-region-specific manner in cerebellum and prefrontal cortex of mouse brain. We found
that Pol II and TF profiles are highly characteristic of the brain regions. The Pol II binding
profiles had 4,218 differential peaks between cerebellum and PFC, while EGR1/MEF2C profiles
had >1,000 differential peaks between the two brain regions. The peaks with their intensity high
in PFC and low in cerebellum were highly enriched in functions including cognition, learning or
memory, while the peaks that were high in cerebellum and low in PFC were enriched in GO
terms including walking behavior, motor coordination and cerebellar cortex formation. The fact
that the binding profiles of these functional molecules are highly characteristic of the functions
of various brain regions indicate that Pol II and the key TFs are critically involved in the
molecular dynamics associated with the spatial configuration of brain functions. Our results
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suggest the possibility of deciphering genome-wide molecular binding profiles to establish the
neuroanatomical origin of brain tissues for the first time.
Methods Cell culture GM12878 cells were obtained from Coriell Institute for Medical Research. Cells were cultured in RPMI-1640 medium (30-2001, ATCC) with 15% fetal bovine serum (16000-044, Gibco) and 1% pen-strep (Invitrogen) at 37°C, 5% CO2. Cells were subcultured every 3 d to maintain exponential growth.
Mouse strain and brain dissection C57BL/6J mice were purchased from Jackson Laboratory and maintained in the animal facility with 12-h light/12-h dark cycles and food and water ad libitum. 8-week old male mice were sacrificed by compressed CO2 followed by cervical dislocation. Mouse brains were rapidly dissected, frozen on dry ice and stored at -80°C. This study was approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Tech.
Nuclei isolation from brain tissues A mouse brain was put on ice and PFC and cerebellum were dissected for nuclei isolation. The following steps are performed on ice and centrifugation performed at 4°C. Tissue was placed in 3 ml of ice-cold nuclei extraction buffer [0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 0.1 mM EDTA, 10 mM tris-HCl, and 0.1% Triton X-100, with 30 µl of PIC (P8340, Sigma-Aldrich), 3 µl of 100 mM PMSF, and 3 µl of 1 M dithiothreitol added before use]. Tissue was homogenized in the grinder set (D9063, Sigma-Aldrich) by slowly douncing 15 times with pestle A and 25 times with pestle B. Homogenate was filtered through a 40 µm cell strainer into a 15 ml tube and centrifuged at 1000g for 10 min. The supernatant was removed and the pellet was resuspended in 500 µl nuclei extraction buffer and transferred to a 1.5 ml tube. 750 µl of 50% iodixanol, 7.5 µl of PIC, 0.75 µl of 100 mM PMSF and 0.75 µl of 1M dithiothreitol were added and mixed by pipetting. The mixture was centrifuged at 10,000g for 20 min and the supernatant was removed. If mixed nuclei (without separation of neurons and glia) were used for MOWChIP directly, the nuclei pellet was resuspended in 200 µl of Dulbecco's phosphate-buffered saline (DPBS). If nuclei labeling and FACS sorting were conducted, 500 µl of 2% normal goat serum (50062Z, Life Technologies) in DPBS was added to the nuclei pellet and incubated for 10 min before resuspending. Anti-NeuN antibody conjugated with Alexa 488 (MAB377X, EMD Millipore) was diluted with DPBS to 2 ng/µl. 8 µl of anti-NeuN was added to each 500 µl of nuclei suspension and incubated at 4°C for 1 h on a rotator. The labeled nuclei were then sorted using FACS (BD FACSAria, BD Biosciences). 8 µl of non-labeled nuclei were saved as unstained control prior to addition of anti-NeuN antobody. The concentration of nuclei suspension after FACS was typically low (~1.2×105/ml). The nuclei were re-concentrated by adding 200 µl of 1.8 M sucrose, 5 µl of 1M CaCl2 and 3 µl of 1M Mg(Ac)2 to 1 ml of the nuclei suspension. The mixture was incubated on ice for 15 min and centrifuged at 1800g for 15 min. Supernatant was removed and the nuclei pellet was resuspended in DPBS.
MNase digestion of chromatin
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This protocol is scalable in volume and can digest cell/nuclei suspension with concentration up to 4×106/ml. Our experiments usually started with 4×105 cells/nuclei suspended in 100 µl of DPBS. 1 µl of PIC, 1 µl of 100 mM PMSF and 100 µl lysis buffer [4% Triton X-100, 100 mM tris-HCl, 100 mM NaCl, and 30 mM MgCl2] were added, mixed by vortexing and incubated at room temperature for 10 min. 10 µl of 100 mM CaCl2 and 2.5 µl 100U MNase (88216, Thermo Fisher Scientific) were added, mixed by vortexing and incubated at room temperature for 10 min. 22 µl of 0.5 M EDTA was then added, mixed by vortexing and incubated on ice for 10 min. The solution was centrifuged at 16,100g for 5 min at 4°C. Supernatant containing fragmented chromatin was collected into a new 1.5 ml tube and placed on ice for use.
Preparation of immunoprecipitation (IP) beads 5 µl of protein A Dynabeads (10001D, Invitrogen) were used in each MOWChIP assay. The beads were washed twice with IP buffer (20 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% (w/v) sodium deoxycholate, 0.1% SDS, 1% (v/v) Triton X-100) and resuspended in 150 µl of IP buffer. 1 µg of Pol II or TF antibody was added into the bead suspension for each assay using 105 cells/nuclei, while 0.5 µg of antibody was added for each assay using 5×104 or fewer cells/nuclei. We used the following antibodies in this work: anti-Pol II-total (ab817, lot GR3271868-2, Abcam), anti-Pol II-S5 (ab5131, lot GR3202335-5, Abcam), anti-EGR1 (sc-101033, lot A1618, Santa Cruz), anti-MEF2C (sc-365862, lot B0818, Santa Cruz), anti-HDAC2 (ab124974, lot GR97402-7, Santa Cruz). The suspension was incubated on a rotator at 4°C for 2 h. The beads were then washed with IP buffer twice and resuspended in 5 µl of IP buffer for loading into the device chamber.
MOWChIP-seq The microfluidics-based MOWChIP-seq process, including microfluidic device fabrication and operation, was conducted following our published protocol14.
Purification of ChIP DNA After MOWChIP, IP beads were rinsed once with IP buffer and resuspended in 200 µl of DNA elution buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM EDTA, and 0.03% SDS). 2 µl of 20 mg/ml proteinase K was added and the suspension was incubated at 65°C for 1 h. DNA was then extracted and purified by phenol-chloroform extraction and ethanol precipitation. DNA pellet was resuspended in 8 µl of low EDTA TE buffer. Input DNA was purified with the same process, by digesting chromatin solution directly using proteinase K, followed by the same extraction and purification process.
Library preparation, quantification and sequencing Libraries were constructed using Accel-NGS 2S Plus DNA Library kit (Swift Biosciences) following the manufacturer’s instructions. 1× EvaGreen dye (Biotium) was added to the amplification mixture to monitor the PCR amplification. Library was eluted to 7 µl of low EDTA TE buffer. Library fragment size was examined using TapeStation (Agilent) and the concentration was quantified with KAPA Library Quantification kit (Kapa Biosystems). We also examined the enrichment of the libraries using qPCR and primers listed in Supplementary Table S3. Libraries were pooled for sequencing by Illumina HiSeq 4000 SR50 mode.
ChIP-seq data analysis
15 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451963; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Raw sequencing FASTQ files were trimmed using Trim Galore! with default settings. Reads that passed the quality check were mapped to reference genomes hg38 (human) or mm10 (mouse) with Bowtie.59 Uniquely mapped reads were filtered for known blacklisted genome regions using Samtools60 and bedtools61 to remove ChIP-seq artifacts. The reference genome was then divided into 100-bp bins and ChIP-seq signal for each bin was counted for both ChIP and input samples. Normalized ChIP-seq signal for each bin was calculated using the following equation: �ℎ�� ������ ����� ������ ������ = ( − ) × 1,000,000 ��. �� ������ �ℎ�� ����� ��. �� ������ ����� ����� Both ChIP and input reads were then extended by 100 bp on either ends to compute normalized signal for each bin in the same manner, and visualized as tracks in genome browser IGV.62 Peak calling was conducted using MACS2 (default settings). Differential binding analysis was carried out using DiffBind56 with default settings. Selected genomic regions of interest were further analyzed for gene ontology terms using GREAT.63 Pearson’s correlation between datasets was calculated via DiffBind, using dba.count command with default parameters to process MACS2 peaks and uniquely mapped reads and calculate consensus peaks and affinity score. ROC curves were created by calculating the true positive rate (TPR) and false positive rate (FPR) at 100 different peak-calling thresholds from 0.99 and 10-15. Thresholds were applied to both the EGR1 and Pol II sets, and AUC was calculated with the R package ROC. Subsampling of EGR1 bam files to between 25% to 95% of original reads was performed using Samtools. Five separate, but consistent, seeds were used at each subsampling percentage. Matthew’s correlation coefficient (MCC) was calculated in R (mltools) at each percentage and each seed using a MACS2 q-value cutoff of 0.05 for both EGR1 and Pol II peaks.
Data Availability The ChIP-seq data sets are deposited in the Gene Expression Omnibus (GEO) repository with the following accession number GSE172224. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE172224
Acknowledgements This work was supported by US National Institutes of Health (NIH) grants R33 CA214176 (C.L.), R01EB017235 (C.L.), R01 CA243249 (C.L.), P30 CA012197 (C.L.), R01NS094574 (H.X.), R21MH120498 (H.X.), and a seed grant from Virginia Tech Institute for Critical Technology and Applied Science (C.L.).
Author Contribution C.L. designed and supervised the study. Z.L. conducted all nMOWChIP-seq experiments and analyzed the data. L.B.N., Y.Z., C.D., Q. Z., B.Z., Z.Z., M.S. helped with data analysis and experiments. A.M. and H.X. helped with experiments on the TFs. Z.L., L.B.N. and C.L. wrote the manuscript. All authors proofread the manuscript and provided feedback.
Competing interests C.L. holds a US patent on MOWChIP-seq. The other authors declare no competing interests.
References
16 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451963; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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20 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451963; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figures and captions
A
MNase digestion Library preparation
Antibody-coated beads ΔP
Capture native RNA Pol II/TF binding without crosslinking MOWChIP Oscillatory washing
100 kb hg38
[0 - 10.00] B GM_Pol 2_50K_1_Full _Norma10 lized _Extended .bwR1 0 50,000 [0 - 10.00] GM_Pol 2_50K_2_Full _Norma10 lized _Extended .bwR2 0 [0 - 10.00] GM_Pol 2_10K_1_Full _Norma10 lized _Extended .bwR1 0 10,000 [0 - 10.00] GM_Pol 2_10K_2_Full _Norma10 lized _Extended .bwR2 0 [0 - 10.00] GM_Pol 2_2K_1_Full _Normal10 Pol II (S5) ized _Extended .bwR1 0 2,000 [0 - 10.00] GM_Pol 2_2K_2_Full _Normal10 ized _Extended .bwR2 0 [0 - 10.00] GM_Pol 2_1K_1_Full _Normal10 ized _Extended .bwR1 0 1,000 [0 - 10.00] GM_Pol 2_1K_2_Full _Normal10 ized _Extended .bwR2 0 [0 - 10.00] ENCODE_GM_Pol 2-4H8_110_Ful l_NormalizedENCODE_Extended .bw 0
RefSeqGene gene ZC3H10 MYL6MYL6 RNF41RNF41 ANKRD52 CSCS CNPY2 IL23AIL23A TIMELESSTIMELESS SPRYD4 SNORA105C RBMS2RBMS2 RBMS2 BAZ2ABAZ2A PTGES3PTGES3 NACANACA HSD17B6
100 kb hg38 [0 - 30] CLu-JE8-GM-EGR1-100 K-1_F30 ull _Normalized _ExtendedR1. bw 0 100,000 [0 - 30] CLu-JE8-GM-EGR1-100 K-2_F30 ull _Normalized _ExtendedR2. bw 0 [0 - 30] CLu-JE8-GM-EGR1-50K-1_Fu30 ll _Normalized _ExtendedR1.b w 0 50,000 [0 - 30] CLu-JE8-GM-EGR1-50K-2_Fu30 ll _Normalized _ExtendedR2.b w 0 [0 - 20] CLu-YP24-GM10K-EGR1-2_Fu20 EGR1 ll _Normalized _ExtendedR1.b w 0 10,000 [0 - 20] CLu-Liu -JE4-GM10K-Egr1-2 20 _Full _Normalized _ExtendeR2 d.bw 0 [0 - 20] CLu-YP24-GM5K-EGR1-1_Ful20 l_Normalized _ExtendedR1.bw 0 5,000 [0 - 20] CLu-Liu -JE4-GM5K-Egr1-1_ 20 Full _Normalized _ExtendedR2 .bw 0 [0 - 3.00] ENCODE_GM_EGR1_1_Full _No3 rmalizedENCODE_Extended .bw 0
GeneRefSeq gene H1-1 H2BC3H2BC3 NR_144383.1 H2AC6H2AC6 H1-4 H2BC6H2BC6 H2BC8 H4C7H4C7 H3C8 XR_241992.4 BTN3A2BTN3A2 BTN3A1 BTN3A3BTN3A3 XR_001744057.2 NR_13461NR_134611.1
100 kb hg38
[0 - 20] GM_HDAC2_100 K_1_R1_20Full _ Normalized _ExtendedR1.bw 0 100,000 [0 - 20] GM_HDAC2_100 K_2_R1_20Full _ Normalized _ExtendedR2.bw 0 [0 - 15] GM_50K_1_R1_Full _Normali15 HDAC2 zed_Extended .bwR1 0 50,000 [0 - 15] GM_50K_2_R1_Full _Normali15 zed_Extended .bwR2 0 [0 - 1.00] ENCODE_GM_HDAC2_1_Full1 _N ormalizedENCODE_Extended .bw 0 Gene RefSeq gene PRSS16 POM121L2POM121L2 ZNF204PZNF204P ZNF184ZNF184 XR_001744071.1XR_001744071.1 LINC01012LINC01012 NR_038929.1 H2AC13H2AC13 H2AC16H2AC16 OR2B2
C CorrelationPol of II peaks (S5) Pol 2-S5 EGR1 HDAC2 Correlation of peaks EGR 1 Correlation of promoters TERC 1.0 1.0 1.01.0 XR150K_R1 X100R1K_R1
50,000 100,000 X100R1K_R1 X50K_R2 0.9 X100K_R2 R2 R2 0.9 0.90.9 100,000 XR110K_R1 X50R1K_R1
10,000 0.50,0008 X100R2K_R2 0.8 0.80.8 XR210K_R2 X50R2K_R2
XR12K_R1 X10R1K_R1 0.7 0.7 0.7 2,000 10,000 X50R1K_R1 0.7 XR22K_R2 X10R2K_R2 50,000 0.6 XR11K_R1 XR15K_R1 0.6 0.60.6 1,000 5,000 X50R2K_R2 XR21K_R2 XR25K_R2
0.5 0.5 0.5 2 1 2 1 2 1 2 1 2 1 2 1 2 1
2 1 0.5 2 1 2 1 R R R R R R R R R R R R R R R R R R R R ______K K K K K K K K K K K K K K K K K K K K 1 1 2 2 5 5 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 10 10 50 50 R2 R1 R2 R1 R2 R1 R2 R1 10 10 50 50 50 50 100 100 100 100 1,000 2,000 5,000 10,000 50,000 10,000 10,000 50,000 50,000 100,000
Fig 1. Overview of the low-input nMOWChIP process and data on RNA polymerase II, EGR1 and HDAC2 binding in GM12878 cells. (A) Steps to generate nMOWChIP-seq libraries. (B) Normalized RNA Pol II (S5) signals generated using 1,000 to 50,000 cells, EGR1 signals generated using 5,000 to 100,000 cells, and HDAC2 signals generated using 50,000 and 100,000 cells. ENCODE data (Pol II: GSM803485; EGR1: GSM803434; HDAC2: GSE105521) are included for comparison. (C) Pearson’s correlations of affinity score calculated from consensus MACS2 peaks and uniquely mapped reads using DiffBind, among samples of various cell numbers for RNA Pol II, EGR1 and HDAC2.
21
bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451963; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A 100 kb hg38
[0 - 10.00] GM_Pol 2-total _50K_1_Full10 _Normalized _ExtendedR1.bw 0 Pol II-total [0 - 10.00] GM_Pol 2-total _50K_2_Full10 _Normalized _ExtendedR2.bw 0 [0 - 10.00] GM_Pol2_50K_1_Full _Norma10 lized _Extended .bwR1 0 Pol II-S5 [0 - 10.00] GM_Pol 2_50K_2_Full _Norma10 lized _Extended .bwR2 0 RefSeqGene gene ZC3H10 MYL6MYL6 RNF41RNF41 ANKRD52 CSCS CNPY2 IL23AIL23A TIMELESSTIMELESS SPRYDSPRYD44 SNORA105C RBMS2RBMS2 RBMS2 BAZ2ABAZ2A PTGES3PTGES3 NACANACA HSD17B6 B 3 kb hg38 20 kb hg38
[0 - [100 -. 0022]] [0 - [100 -. 002213]] GM_Pol 2-total _50K_1_Full 10 GM_Pol 2-total _50K_1_Full 10 _Normalized _Extended .bw R1 _Normalized _ExtendedR1.bw 0 0 Pol[0 - [100II -. 0022-]] total Pol II-total [0 - [100 -. 002213]] GM_Pol 2-total _50K_2_Full 10 GM_Pol 2-total _50K_2_Full 10 _Normalized _Extended .bw R2 _Normalized _ExtendedR2.bw 0 0 [0 - [100 -. 0022]] [0 - [100 -. 002213]] GM_Pol2_50K_1_Full _Norma 10 GM_Pol2_50K_1_Full _Norma10 lized _Extended .bw R1 lized _Extended .bw R1 0 0 Pol[0 - [100 -. 0022II]] -S5 Pol II-S5 [0 - [100 -. 002213]] GM_Pol 2_50K_2_Full _Norma 10 GM_Pol 2_50K_2_Full _Norma10 lized _Extended .bw R2 lized _Extended .bw R2 1.00 0 0 Gene 0.5 Gene ACTG1ACTG1 NR_FSCN0734942 .1 FSCNMDM2MDM2 2 CPM CPM C D 1.00 0.4 0.5 0.75
Legend 0.4 0.75 Legend 0.3 LegendencPol2-S5: 0.871 LegendencPol2-S5 encPol2-S5: 0.871 0.3 encPol2-S5 0.50 encPol2-total : 0.807 encPol2-total tpr 0.50 encPol2-total : 0.807 encPol2-total TPR tpr Mean mean PolPol2-S25-: S0.5899: 0.899 mean PolPol2-S25-S5 0.2 0.2 Pol2-total: 0.845 Pol2-total Pol2-total: 0.845 Pol2-total 0.25 0.1 0.25 0.1
0.00 0.0
0.00 0.25 0.50 0.75 1.00 100 80 60 40 FPRfpr Subsamplesubsample (%) 0.00 0.0 Fig 2. Comparison of Pol II-total and Pol II-S5 bindings in GM12878 cells. (A) Normalized Pol II-S5 and Pol II- 0.00 0.25 0.50 0.75 1.00 100 80 60 40 total signal in GM12878fpr cells (50,000 cells per assay). (B) Normalizedsubsample Pol II signal over genes ACTG1 and MDM2, showing the difference in Pol II-total and Pol II-S5 binding. Pol II-total includes Pol II that is not phosphorylated. These non-active Pol II molecules tend to pause at promoter regions, while phosphorylated and active Pol II (S5 being the majority) binds to the entire gene body. (C) Receiver operating characteristic (ROC) curves using ENCODE (SRX100400 and SRX100530) and nMOWChIP-seq Pol II-total and Pol II S5 datasets predicting EGR1 binding peaks in GM12878 cells. (D) Matthew’s correlation coefficient (MCC) plots of ENCODE and nMOWChIP- seq datasets being subsampled at different levels to predict EGR1 binding peaks in GM12878 cells.
22 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451963; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Cerebellum Cerebellum PFC PFC - - - - 50 50 50 50 K K K K - - - - Pol Pol Pol Pol 2 2 2 2 - - - - total total total total
Correlation of peaks Pol 2-total - -
- - 2 1 2 1
5 .
A B 0 11.0.0
1 . total . 2 Pol . K 50 .
Cerebellum .50K.Pol2.total.2 PFC
6 .
100 kb mm10 R10 00.9.9 [0 - 40] PFC_mix _Pol 2-total _50K_1 40 PFC
_Full _Normalized _Extende
2 . total . 2 Pol . K 50 .
d.bw R1 PFC
7 . 0 Cerebellum .50K.Pol2.total0 .1 R2 00.8.8 [0 - 40] PFC PFC_mix _Pol 2-total _50K_2 40 _Full _Normalized _Extende
d.bw R2
8 .
0 0
1 . total . 2 Pol . K 50 . 0.7 Cerebellum [0 - 40] PFC.50K.Pol2.total.2 0.7 Cerebellum _mix _Pol 2-tota 40 R1 l_50K_1_Full _Normalized _ R1
Extended .bw 0 Cerebellum 9 .
[0 - 40] 0 00.6.6 2 . total . 2 Pol . K 50 . Cerebellum _mix _CerebellumPol 2-tota Cerebellum 40 PFC.50K.Pol2.total.1 l_50K_2_Full _Normalized _ R2 R2
Extended .bw 0
0 .
1 00.5.5 1 2
1 2 - -
- - total - 2 Refseq genes Pol peaks of Correlation GalRefSeq3st3 Cst6geneSart1 Drap1 Fibp Cfl1 Ovol1 Rnaseh2c Rela Pcnx3 Ehbp1l1 Scyl1 Malat1 Neat1 Slc25a45 Pola2 Capn1 Syvn1 Zfpl1 Snx15 Batf2 total total R2 R1 R2 R1 total total
Drap1 Cfl1 Ovol1 Rela Ehbp1l1 Malat1 Slc25a45 - - - - 2 2 2 2 Pol Pol Pol Pol - - - - K K K K 50 50 50 50 PFC - - - - PFC PFC Job ID: 20210112-public-4.0.4-87kaoV Job ID: 20210112-public-4.0.4-87kaoV C Display nam e: m ouse Pol2 all sam ples PFC over Cere_50K.bed GO Biological ProcessDisplay nam e: m ouse Pol2 all sam ples PFC over Cere_50K.bed M ouse Phenot yCerebellum pe SiCerebellum ngle KO PFC > Cerebellum Cerebellum -log10(Binom ial p value) -log10(Binom ial p value) GO biological process GO mouse phenotype single KO 0 2 4 6 8 10121416182022 0 2 4 6 8 1012141618202224 regulation of neuronal synaptic plasticity 22.75 abnorm al object recognition m em ory 25.97 positive regulation of dendritic spine m orphogenesis 22.02 abnorm al long term potentiation 23.50 regulation of synaptic plasticity 21.56 decreased anxiety-related response 20.30 regulation of dendritic spine m orphogenesis 20.43 abnorm al glutam ate-m ediated receptor currents 19.31 regulation of dendrite developm ent 19.72 abnorm al synaptic plasticity 16.21 learning 19.15 reduced long term potentiation 15.32 regulation of dendritic spine developm ent 18.53 abnorm al synaptic depression 15.02 positive regulation of dendrite developm ent 18.49 abnorm al m ean corpuscular volum e 13.87 regulation of ion transm em brane transporter activity 17.79 reduced long term depression 13.80 positive regulation of dendritic spine developm ent 17.07 abnorm al long term depression 13.31 regulation of transporter activity 16.26 enhanced long term potentiation 12.80 regulation of transm em brane transporter activity 16.14 abnorm al excitatory postsynaptic potential 12.79 habituation 15.01 abnorm al hippocam pus pyram idal cell m orphology 12.18 Job ID: 20210112-public-4.0.4-GGsEux regulation of Rho protein signal transduction 14.55 Job ID: 202a10b1n1o2-rpmubalicl -4e.x0.c4i-tGaGtsoEurxy postsynaptic currents 11.84 Display nam e: m ouse Pol2 all sam ples Crerge uovlaert iPoFnC_ 5o0fK R.baeds protein signal transductiGonO Biological 1P4.r29ocess Displaaby nnoamrme:aml oluosne gPo tl2e arlml sa ombpjlesc Cte re ocvoegr nPFiCti_o50nK .mMbeedomuorsy e Phenot1y1.p67e Single KO regulation of calcium ion transport Cerebellum13.71 > PFC abnorm al spatial working m em ory 11.60 -log10(Binom ial p value) -log10(Binom ial p value) regulation GOof d ebiologicalndrite m orp processhogenesis 13.14 prefereGOnce mousefor addic phenotypetive substanc singlee KO 10.91 associative learning0 1 2 3 4 5 6 7 1829.8100111213 abnorm al paired-pulse facilitation0 2 4 6 8 1011021.045161820222426 cerebellar granule cells doicfifaelr ebnethiavtiioonr 11.09 13.30 abnormabanl oarvmoiadla hnicned blerairnn imngo rbpehhoalovigoyr 9.59 26.09 cerebellar granular layer mviosurpahl obgeehnaevsioisr 11.08 12.39 abnorm al cerebellum veirmmpisa imreodr plehaorlnoignyg 9.46 22.46 cerebellar granular layer developm ent 12.07 abnorm al cerebellum m orphology 21.02 cerebellar cortex form ation 12.05 abnorm al cerebellar cortex m orphology 20.03 m odulation of excitatory postsynaptic potential 11.42 cerebellum verm is hypoplasia 19.89 cell differentiation in hindbrain 10.95 abnorm al m etencephalon m orphology 19.54 cerebellar cortex m orphogenesis 10.40 abnorm al hindbrain developm ent 18.98 hindbrain m orphogenesis 9.37 abnorm al forebrain developm ent 15.91 cerebellum m orphogenesis 8.33 abnorm al cerebellar layer m orphology 13.43 cerebellar cortex developm ent 8.16 abnorm al hippocam pus developm ent 12.26 positive regulation of transm em brane transport 6.50 photophobia 12.19 positive regulation of ion transm em brane transporter activity 5.97 abnorm al cerebellum developm ent 12.08 positive regulation of transporter activity 5.67 ataxia 11.46 calcium ion-regulated exocytosis of neurotransm itter 4.95 abnorm al cerebellar foliation 10.46 positive regulation of potassium ion transm em brane transporter activity 4.94 open neural tube 10.15 nDegative regulation of protein serine/threonine kinase activity 4.91 abnorm al capillary branching pattern 9.74 re5s pkbonse to mm10auditory stim ulus 54 kb.39 mm10 10 kb mm10abnorm al eye developm ent 5 kb 9.mm1044 [0 - 40] exocyto[s0 -i 20s] 4.34 [0 - 10.00] [0 - 7.00] aniridia 9.36 PFC_mix _Pol 2-total _50K_1 PFC_mix _Pol 2-total _50K_1 PFC_mix _Pol 2-total _50K_1 PFC_mix _Pol 2-total _50K_1 40 neurotransm itter secretion20 4.00 10 abnorm al cerebellum lobule m7 orphology 9.30 _Full _Normalized _Extende R1 _Full _Normalized _Extende _Full _Normalized _Extende _Full _Normalized _Extende d.bw 0 calcium ion redg.bwulated exocytosis 0 3.81 d.bw 0 d.bw abnorm al spinal cord dorsal horn m0 orphology 9.09 PFC [0 - 40] [0 - 20] [0 - 10.00] [0 - 7.00] PFC_mix _Pol 2-total _50K_2 40 PI=6.6PFC_mix _Pol 2-total _50K_2 20 PI=3.4PFC_mix _Pol 2-total _50K_2 10 PFC_mix _Pol 2-total _50KPI=9.9_2 7 PI=7.9 _Full _Normalized _Extende R2 _Full _Normalized _Extende _Full _Normalized _Extende _Full _Normalized _Extende d.bw 0 d.bw 0 d.bw 0 d.bw 0 [0 - 40] [0 - 20] [0 - 10.00] [0 - 7.00] Cerebellum _mix _Pol 2-tota 40 Cerebellum _mix _Pol 2-tota 20 Cerebellum _mix _Pol 2-tota 10 Cerebellum _mix _Pol 2-tota 7 l_50K_1_Full _Normalized _ R1 l_50K_1_Full _Normalized _ l_50K_1_Full _Normalized _ l_50K_1_Full _Normalized _ Extended .bw 0 Extended .bw 0 Extended .bw 0 Extended .bw 0 Cerebellum [0 - 40] [0 - 20] [0 - 10.00] [0 - 7.00] Cerebellum _mix _Pol 2-tota 40 PI=74.0Cerebellum _mix _Pol 2-tota 20 PI=37.7Cerebellum _mix _Pol 2-tota 10 Cerebellum _mix_Pol2-totaPI=3.2 7 PI=1.1 l_50K_2_Full _Normalized _ R2 l_50K_2_Full _Normalized _ l_50K_2_Full _Normalized _ l_50K_2_Full _Normalized _ Extended .bw 0 Extended .bw 0 Extended .bw 0 Extended .bw 0 RefseqRefSeq genes gene Refseq genes Refseq genes Refseq genes Plk2Plk2 Npas4Npas4 Slc46a1Slc46a1 Mrps18a Sarm1 Rsph9Rsph9 Mad2l1bp
E PFC Cerebellum
9% 7%
No Pol II binding 18% 37% Paused and unexpressed (PI>=20) 43% 27% Paused and expressed (2<=PI<20)
Non-paused and expressed (PI<2)
32%
27%
Fig 3. Differential RNA polymerase II binding in prefrontal cortex and cerebellum of mouse brain. (A) Normalized Pol II-total signals generated using nuclei isolated from mouse prefrontal cortex (PFC) and cerebellum (50,000 nuclei per assay). (B) Pearson’s correlations of affinity score calculated from consensus MACS2 peaks and uniquely mapped reads using DiffBind, among Pol II data on PFC and cerebellum. (C) GO biological processes and mouse phenotypes (single knockout) associated with regions having higher Pol II binding levels in either PFC or cerebellum (-log10 binomial p value, fold change > 2, p < 10-5). (D) Normalized Pol II-total signals at genes that have significantly different pausing indexes between PFC and cerebellum. (E) Distribution of Pol II pausing index in PFC and cerebellum.
23 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451963; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A 200 kb mm10 100 kb mm10 20 kb mm10 [0 - 2.50] [0 - 2.50] [0 - 7.00] [0 - 7.00] PFC_mix _EGR1_100 K_1_Full2.5 PFC_mix _EGR1_100 K_1_Full PFC_mix _EGR1_100 K_1_Full 7 PFC_mix _EGR1_100 K_1_Full _Normalized _ExtendedR1.bw _Normalized _Extended .bw _Normalized _Extended .bw _Normalized _Extended .bw 0 0 PFC EGR1 [0 - 2.50] [0 - 2.50] [0 - 7.00] [0 - 7.00] PFC_mix_EGR1_100K_2_Full2.5 PFC_mix_EGR1_100K_2_Full PFC_mix_EGR1_100K_2_Full 7 PFC_mix_EGR1_100K_2_Full _Normalized _ExtendedR2.bw _Normalized _Extended .bw _Normalized _Extended .bw _Normalized _Extended .bw 0 0 [0 - 2.50] [0 - 2.50] [0 - 7.00] [0 - 7.00] PFC_mix _MEF2C_100 K_1_Ful2.5 PFC_mix _MEF2C_100 K_1_Ful PFC_mix _MEF2C_100 K_1_Ful 7 PFC_mix _MEF2C_100 K_1_Ful l_Normalized _ExtendedR1.bw l_Normalized _Extended .bw l_Normalized _Extended .bw l_Normalized _Extended .bw 0 0 PFC MEF2C [0 - 2.50] [0 - 2.50] [0 - 7.00] [0 - 7.00] PFC_mix _MEF2C_100 K_2_Ful2.5 PFC_mix _MEF2C_100 K_2_Ful PFC_mix _MEF2C_100 K_2_Ful 7 PFC_mix _MEF2C_100 K_2_Ful l_Normalized _ExtendedR2.bw l_Normalized _Extended .bw l_Normalized _Extended .bw l_Normalized _Extended .bw 0 0 [0 - 2.50] [0 - 2.50] [0 - 7.00] [0 - 7.00] Cerebellum _mix _EGR1_100 K2.5 Cerebellum _mix _EGR1_100 K Cerebellum _mix _EGR1_100 K 7 Cerebellum _mix _EGR1_100 K _1_Full _NormalizedR1_Exten _1_Full _Normalized _Exten _1_Full _Normalized _Exten _1_Full _Normalized _Exten ded.bw 0 ded.bw ded.bw 0 ded.bw Cerebellum EGR1 [0 - 2.50] [0 - 2.50] [0 - 7.00] [0 - 7.00] Cerebellum _mix _EGR1_100 K2.5 Cerebellum _mix _EGR1_100 K Cerebellum _mix _EGR1_100 K 7 Cerebellum _mix _EGR1_100 K _2_Full _NormalizedR2_Exten _2_Full _Normalized _Exten _2_Full _Normalized _Exten _2_Full _Normalized _Exten ded .bw 0 ded .bw ded .bw 0 ded .bw [0 - 2.50] [0 - 2.50] [0 - 7.00] [0 - 7.00] Cerebellum _mix _MEF2C_1002.5 Cerebellum _mix _MEF2C_100 Cerebellum _mix _MEF2C_100 7 Cerebellum _mix _MEF2C_100 K_1_Full _NormalizedR1_Exte K_1_Full _Normalized _Exte K_1_Full _Normalized _Exte K_1_Full _Normalized _Exte Cerebellum MEF2Cnded .bw 0 nded .bw nded .bw 0 nded .bw [0 - 2.50] [0 - 2.50] [0 - 7.00] [0 - 7.00] Cerebellum _mix _MEF2C_1002.5 Cerebellum _mix _MEF2C_100 Cerebellum _mix _MEF2C_100 7 Cerebellum _mix _MEF2C_100 K_2_Full _NormalizedR2_Exte K_2_Full _Normalized _Exte K_2_Full _Normalized _Exte K_2_Full _Normalized _Exte nded .bw 0 nded .bw nded .bw 0 nded .bw
Refseq genes Refseq genes Refseq genes Refseq genes
RefSeqCere geneCere PFC PFC
Heg1Cere Cere Itgb5 Kalrn Kalrn KalrnGm32649Kalrn Ropn1 Gria1 Fam114a2 Cacna1a Gm32854 Ier2 NfixGm40546Dand5 GcdhZic1Rtbdn Zic4 Gm29478 PFC PFC Kalrn Gria1 Cacna1a Nfix Zic1 Zic4 - - - - Mef Mef Mef Mef - - - - Egr Egr Egr Egr 2 2 2 2 1 1 c c 1 1 c c
Correlation of peaks TFs ------2 1 2 1 2 1
B 2 1 C 4 .
0 11.0.0
1 . 1 Egr . PFC
PFC R1Cere.Mef2c.2
5 . 0
2 . 1 Egr .
EGR1 PFC 00.9.9 Promoter (<= 1kb) R2Cere.Mef2c.1
PFC EGR1 Promoter (1-2 kb)
1 . c 2 Mef . PFC
6 . 0 Cere.Egr1.2
PFC R1 00.8.8 Promoter (2-3 kb)
2 . c 2 Mef .
MEF2C PFC 5’ UTR
R2Cere.Egr1.1 PFC MEF2C
7 .
0 00.7.7 3’ UTR
1 . 1 Egr . R1PFC.Mef2c.2 Cere 1st exon
Cerebellum Cerebellum EGR1
8 . 0
2 . 1 Egr . Cere Other exon EGR1 0.6 R2PFC.Mef2c.1 0.6
1st intron
1 . c 2 Mef .
Cere Cerebellum MEF2C
9 . R10 PFC.Egr1.2 Other intron Cerebellum 00.5.5
Downstream (<= 300) 2 . c 2 Mef .
MEF2C PFC.Egr1.1 Cere
R2 Distal intergenic 0 .
1 00.4.4 0 25 50 75 100 1 2
1 2 1 2 1 2 ------TFs peaks of Correlation c c 1 1 c c 1 1 2 2 2 2 Egr Egr Egr Egr - - - - R2 R1 R2 R1 R2 R1 R2 R1 Mef Mef Mef Mef - - - - PFC PFC
Cere Cere Percentage (%) PFC PFC Cere Cere PFC PFC EGR1 EGR1 MEF2C MEF2C Job ID: 20210708-public-4.0.4-ZJF1gw Job ID: 20210708-public-4.0.4-ZJF1gw Cerebellum Display nam e: m m 10_EGR1_Cere-over-PFC_2fold-Cerebellum p-less-1E-5.bed GO Biological ProcesDsis_plEayG naRm e1: m m 10_EGR1_Cere-over-PFC_2fold-p-lessM-1Eo-5.buedse Phenot ype Single KO_EGR1 D EGR1 -log10(Binom ial p value) -log10(Binom ial p value) GO biological process GO mouse phenotype single KO 0 1 2 3 4 5 6 7 8 9 101112 0 2 4 6 8 10 12 14 16 18 endom em brane system organization 12.91 ataxia 19.92 establishm ent of organelle localization 11.18 abnorm al m otor coordination/ balance 17.93 organelle localization 10.57 abnorm al locom otor coordination 16.38 calcium ion regulated exocytosis 9.34 abnorm al hippocam pus granule cell layer 14.52 synaptic vesicle exocytosis 8.98 abnorm al tem poral lobe m orphology 12.75 calcium ion-regulated exocytosis of neurotransm itter 8.78 abnorm al hippocam pus m orphology 12.25 secretion by cell 8.57 im paired righting response 10.87 synaptic vesicle transport 8.55 abnorm al hindbrain developm ent 10.79 exocytosis 8.41 abnorm al cerebellum m orphology 10.62 signal release 8.37 abnorm al hippocam pus granule cell m orphology 10.51 synaptic vesicle localization 8.36 abnorm al forebrain developm ent 10.44 establishm ent of vesicle localization 8.32 abnorm al lim bic system m orphology 10.43 m ulticellular organism al signaling 8.29 abnorm al m etencephalon m orphology 10.42 neurotransm itter secretion 8.18 altered righting response 9.63 regulated exocytosis 8.05 abnorm al nervous system electrophysiology 9.54 synaptic vesicle cycle 8.03 abnorm al postural reflex 9.23 vesicle localization 7.96 abnorm al action potential 9.15 adult walking behavior 7.69 abnorm al cerebellar cortex m orphology 9.14 walking behavior 7.64 abnorm al hippocam pus developm ent 8.96 Job ID:p2r0e2s1y07n0a8p-ptuibcli cp-4r.o0.c4e-ksnsR aiLnj volved in chem ical synaptic transm ission 7.62 Job ID:a2b0n2o10r7m08a-pl ucbelicr-e4b.0e.4l-lkunmRa Ldj evelopm ent 8.78 Display nam e: m m 10_MEF2C_Cere-over-PFC_2fold-p-less-1E-5.bed GO Biological Process_M EDFis2plaCy na m e: m m 10_MEF2C_CereM-oveor-PuFCs_2efold -Pp-lhesse-1En-5o.betdype Single KO_M EF2 C MEF2C -log10(Binom ial p value) -log10(Binom ial p value) GO biological process GO mouse phenotype single KO 0 1 2 3 4 5 6 7 8 9 101112 0.00.51.01.52.02.53.03.54.04.55.05.5 ncRNA processing 12.63 abnorm al paired-pulse facilitation 5.79 ncRNA m etabolic process 10.68 im paired lim b coordination 4.90 rRNA processing 7.06 decreased synaptic depression 4.69 rRNA m etabolic process 6.51 m itochondrial translation 5.96 m RNA transport 5.07 tRNA m odification 4.92 RNA localization 4.88 7-m ethylguanosine RNA capping 4.87 m itochondrial electron transport, NADH to ubiquinone 4.73 RNA transport 4.60 establishm ent of RNA localization 4.51 RNA secondary structure unwinding 4.21 nuclear-transcribed m RNA catabolic process, nonsense-m ediated decay 3.54 cerebellar Purkinje cell layer developm ent 3.52 Fig 4. Differential transcription factor binding in prefrontal cortex and cerebellum of mouse brain. (A) Normalized EGR1 and MEF2C signals at genes identified by DiffBind to have significantly different binding between PFC and cerebellum (fold change > 2, p < 10-5). (B) Pearson’s correlations of affinity score calculated from consensus MACS2 peaks and uniquely mapped reads using DiffBind, among TF data on PFC and cerebellum. (C) Distribution of TF binding peaks (p < 10-5) in various genomic regions. Each peak set is the overlapping peaks from 2 replicates of the same sample. (D) GO biological process and mouse phenotype (single knockout) terms associated with regions having higher EGR1 and MEF2C binding levels in cerebellum than PFC (-log10 binomial p value, fold change > 2, p < 10-5).
24