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Muroy et al. Neuroimmunol Neuroinflammation 2020;7:166-82 I http://dx.doi.org/10.20517/2347-8659.2020.16 Page 175

Loss of Phf15 affects global expression of genes involved in antiviral responses and regulation
of inflammatory processes
To examine global transcriptional changes as a result of Phf15 deletion in microglia, we carried out RNA-
sequencing (RNA-seq) on Phf15 KO SIM-A9 cells under no stimulation conditions and 6 h post LPS
stimulation. We chose to examine the no stimulation condition (0-h time point) based on our KD and KO
time course results showing that baseline is one of the most consistently and strongly deregulated time
points. Importantly, elevated or “leaky” proinflammatory mediator expression at baseline might result in
chronic inflammation leading to neurodegeneration. Similarly, 6 h after LPS stimulation corresponded to
the peak of the transcriptional inflammatory response, with large increases in magnitude for both IL-1 β
and Nos2.

Differential gene expression analysis revealed that 466 genes with log -fold change > 1.5 and P adj < 0.01 were
2
upregulated and 309 genes with log -fold change < -1.5 and P adj < 0.05 were downregulated [Figure 5A].
2
[32]
Biological theme enrichment analysis using Metascape on the upregulated genes revealed that the most
enriched biological process categories under basal conditions were “response to virus” and “cytokine
production” [Figure 5B and C]. Under the “response to virus” category, there was significant upregulation
of various interferon-stimulated genes (ISGs), for example Isg15, interferon induced protein with
tetratricopeptide repeats 1 (Ifit1), Ifit3, interferon regulatory factor 7 (Irf7), 2’-5’-oligoadenylate synthetase 2
(Oas2), and Oas-like 2 (Oasl2) [Figure 5C]. The downregulated genes showed more variability in the types
of pathways affected, largely involving growth, differentiation, and glial cell migration processes [Figure 5A
and Supplementary Figure 8A].

Motif analysis for transcription factor binding sites enriched in the promoters of the upregulated genes
at baseline revealed consensus motifs for interferon regulatory factors (IRFs), i.e., the interferon (IFN)
stimulated response element (ISRE) and motifs for IRF3 and IRF8 specifically in the top 5 best matches.
Activator protein 1 (AP-1) and NF-κB p65 subunit (NF-κB-p65) motifs were also enriched. Both can
regulate expression of canonical proinflammatory cytokines such as TNFα and IL-1β [44,45] [Figure 5D].
Motif enrichment for the set of downregulated genes revealed motifs for twist-related protein 2 (Twist2)
and Class A basic helix-loop-helix protein 15 (BHLHA15). Twist2 has been shown to mediate cytokine
downregulation after chronic nucleotide-binding oligomerization domain-containing protein 2 (NOD2,
[46]
a bacterial peptidoglycan sensor) stimulation . BHLHA15 has been shown to induce and maintain
[47]
secretory architecture in cells specialized for secretion [Supplementary Figure 8B].
Differential gene expression analysis after 6 h of LPS stimulation in KO versus control cells revealed 576
upregulated genes (log -fold change > 1.5 and P adj < 0.01) and 322 downregulated genes (log -fold change <
2
2
-1.5 and P adj < 0.05) [Figure 6A]. Interestingly, by 6 h after LPS administration, some of the most enriched
biological process categories in KO cells were related to “cytokine secretion” and “immunoregulatory
interaction” [Figure 6B, C], denoting a strong increase in magnitude of expression of genes involved in
regulating the secretion of proinflammatory mediators. The downregulated genes at 6 h after LPS stimulation
in KO cells relative to control again displayed more variability, but did show decreases in biological process
categories related to “regulation of defense response” and “cytokine production”, indicating negative
regulation of these processes in Phf15 KO cells compared to control [Supplementary Figure 9A].

Motif enrichment analysis for transcription factor binding sites enriched in the promoters of upregulated
genes at the 6-h time point revealed consensus sequences for AP-1, a key regulator of microglia reactivity
[48]
in inflammation [Figure 6D]. Motif enrichment for the set of downregulated genes revealed motifs for
IRFs (the ISRE) and motifs for IRF1 and IRF3 specifically [Supplementary Figure 9B], supporting the
observation that there is a negative “regulation of defense response” by 6 h post stimulation. It is interesting
to note that a functional transition from cytokine production to cytokine secretion seems to occur in the
6-h period after LPS activation.